CRISPR/Cas screening platform to identify genetic modifiers of tau seeding or aggregation

ABSTRACT

Cas-protein-ready tau biosensor cells, CRISPR/Cas synergistic activation mediator (SAM)-ready tau biosensor cells, and methods of making and using such cells to screen for genetic modifiers of tau seeding or aggregation are provided. Reagents and methods for sensitizing such cells to tau seeding activity or tau aggregation or for causing tau aggregation are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 62/820,086,filed Mar. 18, 2019, which is herein incorporated by reference in itsentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The Sequence Listing written in file 544670SEQLIST.txt is 75.7kilobytes, was created on Mar. 17, 2020, and is hereby incorporated byreference.

BACKGROUND

Abnormal aggregation or fibrillization of proteins is a defining featureof many diseases, notably including a number of neurodegenerativediseases such as Alzheimer's disease (AD), Parkinson's disease (PD),frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS),chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease (CJD),and others. In many of these diseases, the fibrillization of certainproteins into insoluble aggregates is not only a hallmark of disease,but has also been implicated as a causative factor of neurotoxicity.Furthermore, these diseases are characterized by propagation ofaggregate pathology through the central nervous system followingstereotypical patterns, a process which correlates with diseaseprogression. The identification of genes and genetic pathways thatmodify the processes of abnormal protein aggregation, or cell-to-cellpropagation of aggregates, are therefore of great value in betterunderstanding neurodegenerative disease etiology as well as in devisingstrategies for therapeutic intervention.

SUMMARY

Provided herein are methods of screening for genetic modifiers of tauaggregation, methods of producing a conditioned medium for inducing orsensitizing to tau aggregation, and methods of generating a populationof tau-aggregation-positive cells. Also provided herein are Cas-taubiosensor cells or populations of such cells and in vitro cultures ofCas-tau biosensor cells and conditioned medium. Also provided herein areCRISPR/Cas synergistic activation mediator (SAM)-tau biosensor cells orpopulations of such cells and in vitro cultures of SAM-tau biosensorcells and conditioned medium.

In one aspect, provided are methods of screening for genetic modifiersof tau aggregation. Some such methods (CRISPRn) can comprise: (a)providing a population of cells comprising a Cas protein, a first taurepeat domain linked to a first reporter, and a second tau repeat domainlinked to a second reporter; (b) introducing into the population ofcells a library comprising a plurality of unique guide RNAs that targeta plurality of genes; (c) culturing the population of cells to allowgenome editing and expansion, wherein the plurality of unique guide RNAsform complexes with the Cas protein, and the Cas protein cleaves theplurality of genes resulting in knockout of gene function to produce agenetically modified population of cells; (d) contacting the geneticallymodified population of cells with a tau seeding agent to produce aseeded population of cells; (e) culturing the seeded population of cellsto allow tau aggregates to form, wherein aggregates of the first taurepeat domain and the second tau repeat domain form in a subset of theseeded population of cells to produce an aggregation-positive populationof cells; and (f) determining abundance of each of the plurality ofunique guide RNAs in the aggregation-positive population of cellsidentified in step (e) relative to the genetically modified populationof cells in step (c), wherein enrichment of a guide RNA in theaggregation-positive population of cells identified in step (e) relativeto the cultured population of cells in step (c) indicates that the genetargeted by the guide RNA is a genetic modifier of tau aggregation,wherein disruption of the gene targeted by the guide RNA enhances tauaggregation, or is a candidate genetic modifier of tau aggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to enhance tauaggregation.

In some such methods, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation.

In some such methods, the first tau repeat domain and/or the second taurepeat domain comprises a tau four-repeat domain. In some such methods,the first tau repeat domain and/or the second tau repeat domaincomprises SEQ ID NO: 11. In some such methods, the first tau repeatdomain and the second tau repeat domain are the same. In some suchmethods, the first tau repeat domain and the second tau repeat domainare the same and each comprises tau four-repeat domain comprising a tauP301S mutation.

In some such methods, first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such methods, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. Optionally, the Cas proteincomprises SEQ ID NO: 21. Optionally, the Cas protein is encoded by acoding sequence comprising the sequence set forth in SEQ ID NO: 22.

In some such methods, the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter are stably expressed in the population of cells. Insome such methods, nucleic acids encoding the Cas protein, the first taurepeat domain linked to the first reporter, and the second tau repeatdomain linked to the second reporter are genomically integrated in thepopulation of cells.

In some such methods, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells.

In some such methods, the plurality of unique guide RNAs are introducedat a concentration selected such that a majority of the cells receiveonly one of the unique guide RNAs. In some such methods, the pluralityof unique guide RNAs target 100 or more genes, 1000 or more genes, or10000 or more genes. In some such methods, the library is a genome-widelibrary.

In some such methods, a plurality of target sequences are targeted onaverage in each of the targeted plurality of genes. Optionally, at leastthree target sequences are targeted on average in each of the targetedplurality of genes. Optionally, about three to about six targetsequences (e.g., about three, about four, or about six) are targeted onaverage in each of the targeted plurality of genes.

In some such methods, each guide RNA targets a constitutive exon.Optionally, each guide RNA targets a 5′ constitutive exon. In some suchmethods, each guide RNA targets a first exon, a second exon, or a thirdexon.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells by viral transduction. Optionally, each ofthe plurality of unique guide RNAs is in a separate viral vector.Optionally, the plurality of unique guide RNAs are introduced into thepopulation of cells by lentiviral transduction. In some such methods,the population of cells is infected at a multiplicity of infection ofless than about 0.3.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells together with a selection marker, and step(b) further comprises selecting cells that comprise the selectionmarker. Optionally, the selection marker imparts resistance to a drug.Optionally, the selection marker imparts resistance to puromycin orzeocin. Optionally, the selection marker is selected from neomycinphosphotransferase, hygromycin B phosphotransferase,puromycin-N-acetyltransferase, and blasticidin S deaminase. Optionally,the selection marker is selected from neomycin phosphotransferase,hygromycin B phosphotransferase, puromycin-N-acetyltransferase,blasticidin S deaminase, and bleomycin resistance protein.

In some such methods, the population of cells into which the pluralityof unique guide RNAs are introduced in step (b) comprises greater thanabout 300 cells per unique guide RNA.

In some such methods, step (c) is about 3 days to about 9 days.Optionally, step (c) is about 6 days.

In some such methods, step (d) comprises culturing the geneticallymodified population of cells in the presence of conditioned mediumharvested from cultured tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state. Optionally, theconditioned medium was harvested after being on confluenttau-aggregation-positive cells for about 1 to about 7 days. Optionally,the conditioned medium was harvested after being on confluenttau-aggregation-positive cells for about 4 days. Optionally, step (d)comprises culturing the genetically modified population of cells inabout 75% conditioned medium and about 25% fresh medium. In some suchmethods, the genetically modified population of cells is not co-culturedwith the tau-aggregation-positive cells in which a tau repeat domainstably presents in an aggregated state.

In some such methods, step (e) is about 2 days to about 6 days.Optionally, step (e) is about 4 days. In some such methods, the firstreporter and the second reporter are a fluorescence resonance energytransfer (FRET) pair, and the aggregation-positive population of cellsin step (e) is identified by flow cytometry.

In some such methods, abundance is determined by next-generationsequencing. In some such methods, a guide RNA is considered enriched ifthe abundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to thecultured population of cells in step (c).

In some such methods, step (f) comprises determining abundance of eachof the plurality of unique guide RNAs in the aggregation-positivepopulation of cells in step (e) relative to the cultured population ofcells in step (c) at a first time point in step (c) and/or a second timepoint in step (c). Optionally, the first time point in step (c) is at afirst passage of culturing the population of cells, and the second timepoint is in the middle of culturing the population of cells to allowgenome editing and expansion. Optionally, the first time point in step(c) is after about three days of culturing, and the second time point instep (c) is after about six days of culturing.

In some such methods, a gene is considered a genetic modifier of tauaggregation, wherein disruption of the gene enhances tau aggregation (ora candidate genetic modifier of tau aggregation, wherein disruption ofthe gene is expected to enhance tau aggregation), if: (1) the abundanceof a guide RNA targeting the gene relative to the total population ofthe plurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to thecultured population of cells in step (c) at both the first time point instep (c) and the second time point in step (c); and/or (2) the abundanceof at least two unique guide RNAs targeting the gene relative to thetotal population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-positive population of cells in step(e) relative to the cultured population of cells in step (c) at eitherthe first time point in step (c) or the second time point in step (c).

In some such methods, the following steps are taken in step (f) toidentify a gene as a genetic modifier of tau aggregation, whereindisruption of the gene enhances tau aggregation (or a candidate geneticmodifier of tau aggregation, wherein disruption of the gene is expectedto enhance tau aggregation): (1) identifying which of the plurality ofunique guide RNAs are present in the aggregation-positive population ofcells produced in step (e); (2) calculating the random chance of theguide RNAs identified in step (f)(1) being present using the formulanCn′*(x−n′)C(m−n)/xCm, wherein x is the variety of unique guide RNAsintroduced into the population of cells in step (b), wherein m is thevariety of unique guide RNAs identified in step (f)(1), wherein n is thevariety of unique guide RNAs introduced into the population of cells instep (b) that target the gene, and wherein n′ is the variety of uniqueguide RNAs identified in step (f)(1) that target the gene; (3)calculating average enrichment scores for the guide RNAs identified instep (f)(1), wherein the enrichment score for a guide RNA is therelative abundance of the guide RNA in the aggregation-positivepopulation of cells produced in step (e) divided by the relativeabundance of the guide RNA in the cultured population of cells in step(c), and wherein relative abundance is the read count of the guide RNAdivided by the read count of the total population of the plurality ofunique guide RNAs; and (4) selecting the gene if a guide RNA targetingthe gene is significantly below the random chance of being present andabove a threshold enrichment score.

Some such methods (CRISPRa) can comprise: (a) providing a population ofcells comprising a chimeric Cas protein comprising a nuclease-inactiveCas protein fused to one or more transcriptional activation domains, achimeric adaptor protein comprising an adaptor protein fused to one ormore transcriptional activation domains, a first tau repeat domainlinked to a first reporter, and a second tau repeat domain linked to asecond reporter; (b) introducing into the population of cells a librarycomprising a plurality of unique guide RNAs that target a plurality ofgenes; (c) culturing the population of cells to allow transcriptionalactivation and expansion, wherein the plurality of unique guide RNAsform complexes with the chimeric Cas protein and the chimeric adaptorprotein, and the complexes activate transcription of the plurality ofgenes resulting in increased gene expression to produce a geneticallymodified population of cells; (d) contacting the genetically modifiedpopulation of cells with a tau seeding agent to produce a seededpopulation of cells; (e) culturing the seeded population of cells toallow tau aggregates to form, wherein aggregates of the first tau repeatdomain and the second tau repeat domain form in a subset of the seededpopulation of cells to produce an aggregation-positive population ofcells; and (f) determining abundance of each of the plurality of uniqueguide RNAs in the aggregation-positive population of cells identified instep (e) relative to the genetically modified population of cells instep (c), wherein enrichment of a guide RNA in the aggregation-positivepopulation of cells identified in step (e) relative to the culturedpopulation of cells in step (c) indicates that the gene targeted by theguide RNA is a genetic modifier of tau aggregation, whereintranscriptional activation of the gene targeted by the guide RNAenhances tau aggregation, or is a candidate genetic modifier of tauaggregation (e.g., for further testing via secondary screens), whereintranscriptional activation of the gene targeted by the guide RNA isexpected to enhance tau aggregation.

In some such methods, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation.

In some such methods, the first tau repeat domain and/or the second taurepeat domain comprises a tau four-repeat domain. In some such methods,the first tau repeat domain and/or the second tau repeat domaincomprises SEQ ID NO: 11. In some such methods, the first tau repeatdomain and the second tau repeat domain are the same. In some suchmethods, the first tau repeat domain and the second tau repeat domainare the same and each comprises tau four-repeat domain comprising a tauP301S mutation.

In some such methods, first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such methods, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. In some such methods, thechimeric Cas protein comprises the nuclease-inactive Cas protein fusedto a VP64 transcriptional activation domain, optionally wherein thechimeric Cas protein comprises from N-terminus to C-terminus: thenuclease-inactive Cas protein; a nuclear localization signal; and theVP64 transcriptional activator domain. In some such methods, the adaptorprotein is an MS2 coat protein, and wherein the one or moretranscriptional activation domains in the chimeric adaptor proteincomprise a p65 transcriptional activation domain and an HSF1transcriptional activation domain, optionally wherein the chimericadaptor protein comprises from N-terminus to C-terminus: the MS2 coatprotein; a nuclear localization signal; the p65 transcriptionalactivation domain; and the HSF1 transcriptional activation domain. Insome such methods, the chimeric Cas protein comprises SEQ ID NO: 36,optionally wherein the chimeric Cas protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 38. In somesuch methods, the chimeric adaptor protein comprises SEQ ID NO: 37,optionally wherein the chimeric adaptor protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 39.

In some such methods, the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the population of cells. In some such methods, nucleicacids encoding the chimeric Cas protein, the chimeric adaptor protein,the first tau repeat domain linked to the first reporter, and the secondtau repeat domain linked to the second reporter are genomicallyintegrated in the population of cells.

In some such methods, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells.

In some such methods, the plurality of unique guide RNAs are introducedat a concentration selected such that a majority of the cells receiveonly one of the unique guide RNAs. In some such methods, the pluralityof unique guide RNAs target 100 or more genes, 1000 or more genes, or10000 or more genes. In some such methods, the library is a genome-widelibrary.

In some such methods, a plurality of target sequences are targeted onaverage in each of the targeted plurality of genes. Optionally, at leastthree target sequences are targeted on average in each of the targetedplurality of genes. Optionally, about three to about six targetsequences (e.g., about three, about four, or about six) are targeted onaverage in each of the targeted plurality of genes. Optionally, aboutthree target sequences are targeted on average in each of the targetedplurality of genes.

In some such methods, each guide RNA targets a guide RNA target sequencewithin 200 bp upstream of a transcription start site. In some suchmethods, each guide RNA comprises one or more adaptor-binding elementsto which the chimeric adaptor protein can specifically bind. Optionally,each guide RNA comprises two adaptor-binding elements to which thechimeric adaptor protein can specifically bind. Optionally, a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. Optionally, theadaptor-binding element comprises the sequence set forth in SEQ ID NO:33. In some such methods, wherein each of one or more guide RNAs is asingle guide RNA comprising a CRISPR RNA (crRNA) portion fused to atransactivating CRISPR RNA (tracrRNA) portion, and the first loop is thetetraloop corresponding to residues 13-16 of SEQ ID NO: 17, and thesecond loop is the stem loop 2 corresponding to residues 53-56 of SEQ IDNO: 17.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells by viral transduction. Optionally, each ofthe plurality of unique guide RNAs is in a separate viral vector.Optionally, the plurality of unique guide RNAs are introduced into thepopulation of cells by lentiviral transduction. In some such methods,the population of cells is infected at a multiplicity of infection ofless than about 0.3.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells together with a selection marker, and step(b) further comprises selecting cells that comprise the selectionmarker. Optionally, the selection marker imparts resistance to a drug.Optionally, the selection marker imparts resistance to puromycin orzeocin. Optionally, the selection marker is selected from neomycinphosphotransferase, hygromycin B phosphotransferase,puromycin-N-acetyltransferase, and blasticidin S deaminase. Optionally,the selection marker is selected from neomycin phosphotransferase,hygromycin B phosphotransferase, puromycin-N-acetyltransferase,blasticidin S deaminase, and bleomycin resistance protein.

In some such methods, the population of cells into which the pluralityof unique guide RNAs are introduced in step (b) comprises greater thanabout 300 cells per unique guide RNA.

In some such methods, step (c) is about 3 days to about 9 days.Optionally, step (c) is about 6 days.

In some such methods, step (d) comprises culturing the geneticallymodified population of cells in the presence of conditioned mediumharvested from cultured tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state. Optionally, theconditioned medium was harvested after being on confluenttau-aggregation-positive cells for about 1 to about 7 days. Optionally,the conditioned medium was harvested after being on confluenttau-aggregation-positive cells for about 4 days. Optionally, step (d)comprises culturing the genetically modified population of cells inabout 75% conditioned medium and about 25% fresh medium. In some suchmethods, the genetically modified population of cells is not co-culturedwith the tau-aggregation-positive cells in which a tau repeat domainstably presents in an aggregated state.

In some such methods, step (e) is about 2 days to about 6 days.Optionally, step (e) is about 4 days. In some such methods, the firstreporter and the second reporter are a fluorescence resonance energytransfer (FRET) pair, and the aggregation-positive population of cellsin step (e) is identified by flow cytometry.

In some such methods, abundance is determined by next-generationsequencing. In some such methods, a guide RNA is considered enriched ifthe abundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to thecultured population of cells in step (c).

In some such methods, step (f) comprises determining abundance of eachof the plurality of unique guide RNAs in the aggregation-positivepopulation of cells in step (e) relative to the cultured population ofcells in step (c) at a first time point in step (c) and/or a second timepoint in step (c). Optionally, the first time point in step (c) is at afirst passage of culturing the population of cells, and the second timepoint is in the middle of culturing the population of cells to allowgenome editing and expansion. Optionally, the first time point in step(c) is after about three days of culturing, and the second time point instep (c) is after about six days of culturing.

In some such methods, a gene is considered a genetic modifier of tauaggregation, wherein transcriptional activation of the gene enhances tauaggregation (or a candidate genetic modifier of tau aggregation, whereintranscriptional activation of the gene is expected to enhance tauaggregation), if: (1) the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold higher in the aggregation-positive population ofcells in step (e) relative to the cultured population of cells in step(c) at both the first time point in step (c) and the second time pointin step (c); and/or (2) the abundance of at least two unique guide RNAstargeting the gene relative to the total population of the plurality ofunique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to thecultured population of cells in step (c) at either the first time pointin step (c) or the second time point in step (c).

In some such methods, the following steps are taken in step (f) toidentify a gene as a genetic modifier of tau aggregation, whereintranscriptional activation of the gene enhances tau aggregation (or acandidate genetic modifier of tau aggregation, wherein transcriptionalactivation of the gene is expected to enhance tau aggregation): (1)identifying which of the plurality of unique guide RNAs are present inthe aggregation-positive population of cells produced in step (e); (2)calculating the random chance of the guide RNAs identified in step(f)(1) being present using the formula nCn′*(x−n′)C(m−n)/xCm, wherein xis the variety of unique guide RNAs introduced into the population ofcells in step (b), wherein m is the variety of unique guide RNAsidentified in step (f)(1), wherein n is the variety of unique guide RNAsintroduced into the population of cells in step (b) that target thegene, and wherein n′ is the variety of unique guide RNAs identified instep (f)(1) that target the gene; (3) calculating average enrichmentscores for the guide RNAs identified in step (f)(1), wherein theenrichment score for a guide RNA is the relative abundance of the guideRNA in the aggregation-positive population of cells produced in step (e)divided by the relative abundance of the guide RNA in the culturedpopulation of cells in step (c), and wherein relative abundance is theread count of the guide RNA divided by the read count of the totalpopulation of the plurality of unique guide RNAs; and (4) selecting thegene if a guide RNA targeting the gene is significantly below the randomchance of being present and above a threshold enrichment score.

In another aspect, provided are additional methods of screening forgenetic modifiers of tau aggregation. Some such methods (CRISPRn) cancomprise: (a) providing a population of cells comprising a Cas protein,a first tau repeat domain linked to a first reporter, and a second taurepeat domain linked to a second reporter; (b) introducing into thepopulation of cells a library comprising a plurality of unique guideRNAs that target a plurality of genes; (c) culturing the population ofcells to allow genome editing and expansion, wherein the plurality ofunique guide RNAs form complexes with the Cas protein, and the Casprotein cleaves the plurality of genes resulting in knockout of genefunction to produce a genetically modified population of cells; (d)contacting the genetically modified population of cells with a tauseeding agent to produce a seeded population of cells; (e) culturing theseeded population of cells to allow tau aggregates to form, whereinaggregates of the first tau repeat domain and the second tau repeatdomain form in a first subset of the seeded population of cells toproduce an aggregation-positive population of cells and do not form in asecond subset of the seeded population of cells to produce anaggregation-negative population of cells; and (f) determining abundanceof each of the plurality of unique guide RNAs in theaggregation-positive population of cells identified in step (e) relativeto the aggregation-negative population of cells identified in step (e)and/or the seeded population of cells in step (d), and/or determiningabundance of each of the plurality of unique guide RNAs in theaggregation-negative population of cells identified in step (e) relativeto the aggregation-positive population of cells identified in step (e)and/or the seeded population of cells in step (d), wherein enrichment ofa guide RNA in the aggregation-negative population of cells identifiedin step (e) relative to the aggregation-positive population of cellsidentified in step (e) and/or the seeded population of cells in step (d)or wherein depletion of a guide RNA in the aggregation-positivepopulation of cells identified in step (e) relative to theaggregation-negative population of cells identified in step (e) and/orthe seeded population of cells in step (d) indicates that the genetargeted by the guide RNA is a genetic modifier of tau aggregation,wherein disruption of the gene targeted by the guide RNA prevents tauaggregation, or is a candidate genetic modifier of tau aggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to prevent tauaggregation, and/or wherein enrichment of a guide RNA in theaggregation-positive population of cells identified in step (e) relativeto the aggregation-negative population of cells identified in step (e)and/or the seeded population of cells in step (d) or wherein depletionof a guide RNA in the aggregation-negative population of cellsidentified in step (e) relative to the aggregation-positive populationof cells identified in step (e) and/or the seeded population of cells instep (d) indicates that the gene targeted by the guide RNA is a geneticmodifier of tau aggregation, wherein disruption of the gene targeted bythe guide RNA promotes or enhances tau aggregation, or is a candidategenetic modifier of tau aggregation (e.g., for further testing viasecondary screens), wherein disruption of the gene targeted by the guideRNA is expected to promote or enhance tau aggregation.

In some such methods, the Cas protein is a Cas9 protein. Optionally, Casprotein is Streptococcus pyogenes Cas9. In some such methods, the Casprotein comprises SEQ ID NO: 21, optionally wherein the Cas protein isencoded by a coding sequence comprising the sequence set forth in SEQ IDNO: 22.

In some such methods, the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter are stably expressed in the population of cells. Insome such methods, nucleic acids encoding the Cas protein, the first taurepeat domain linked to the first reporter, and the second tau repeatdomain linked to the second reporter are genomically integrated in thepopulation of cells.

In some such methods, each guide RNA targets a constitutive exon.Optionally, each guide RNA targets a 5′ constitutive exon. In some suchmethods, each guide RNA targets a first exon, a second exon, or a thirdexon.

Some such methods (CRISPRa) comprise: (a) providing a population ofcells comprising a chimeric Cas protein comprising a nuclease-inactiveCas protein fused to one or more transcriptional activation domains, achimeric adaptor protein comprising an adaptor protein fused to one ormore transcriptional activation domains, a first tau repeat domainlinked to a first reporter, and a second tau repeat domain linked to asecond reporter; (b) introducing into the population of cells a librarycomprising a plurality of unique guide RNAs that target a plurality ofgenes; (c) culturing the population of cells to allow transcriptionalactivation and expansion, wherein the plurality of unique guide RNAsform complexes with the chimeric Cas protein and the chimeric adaptorprotein, and the complexes activate transcription of the plurality ofgenes resulting in increased gene expression to produce a geneticallymodified population of cells; (d) contacting the genetically modifiedpopulation of cells with a tau seeding agent to produce a seededpopulation of cells; (e) culturing the seeded population of cells toallow tau aggregates to form, wherein aggregates of the first tau repeatdomain and the second tau repeat domain form in a first subset of theseeded population of cells to produce an aggregation-positive populationof cells and do not form in a second subset of the seeded population ofcells to produce an aggregation-negative population of cells; and (f)determining abundance of each of the plurality of unique guide RNAs inthe aggregation-positive population of cells identified in step (e)relative to the aggregation-negative population of cells identified instep (e) and/or the seeded population of cells in step (d), and/ordetermining abundance of each of the plurality of unique guide RNAs inthe aggregation-negative population of cells identified in step (e)relative to the aggregation-positive population of cells identified instep (e) and/or the seeded population of cells in step (d), whereinenrichment of a guide RNA in the aggregation-negative population ofcells identified in step (e) relative to the aggregation-positivepopulation of cells identified in step (e) and/or the seeded populationof cells in step (d) or wherein depletion of a guide RNA in theaggregation-positive population of cells identified in step (e) relativeto the aggregation-negative population of cells identified in step (e)and/or the seeded population of cells in step (d) indicates that thegene targeted by the guide RNA is a genetic modifier of tau aggregation,wherein transcriptional activation of the gene targeted by the guide RNAprevents tau aggregation, or is a candidate genetic modifier of tauaggregation (e.g., for further testing via secondary screens), whereintranscriptional activation of the gene targeted by the guide RNA isexpected to prevent tau aggregation, and/or wherein enrichment of aguide RNA in the aggregation-positive population of cells identified instep (e) relative to the aggregation-negative population of cellsidentified in step (e) and/or the seeded population of cells in step (d)or wherein depletion of a guide RNA in the aggregation-negativepopulation of cells identified in step (e) relative to theaggregation-positive population of cells identified in step (e) and/orthe seeded population of cells in step (d) indicates that the genetargeted by the guide RNA is a genetic modifier of tau aggregation,wherein transcriptional activation of the gene targeted by the guide RNApromotes or enhances tau aggregation, or is a candidate genetic modifierof tau aggregation (e.g., for further testing via secondary screens),wherein transcriptional activation of the gene targeted by the guide RNAis expected to promote or enhance tau aggregation.

In some such methods, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. In some such methods, thechimeric Cas protein comprises the nuclease-inactive Cas protein fusedto a VP64 transcriptional activation domain, optionally wherein thechimeric Cas protein comprises from N-terminus to C-terminus: thenuclease-inactive Cas protein; a nuclear localization signal; and theVP64 transcriptional activator domain. In some such methods, the adaptorprotein is an MS2 coat protein, and wherein the one or moretranscriptional activation domains in the chimeric adaptor proteincomprise a p65 transcriptional activation domain and an HSF1transcriptional activation domain, optionally wherein the chimericadaptor protein comprises from N-terminus to C-terminus: the MS2 coatprotein; a nuclear localization signal; the p65 transcriptionalactivation domain; and the HSF1 transcriptional activation domain. Insome such methods, the chimeric Cas protein comprises SEQ ID NO: 36,optionally wherein the chimeric Cas protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 38. In somesuch methods, the chimeric adaptor protein comprises SEQ ID NO: 37,optionally wherein the chimeric adaptor protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 39.

In some such methods, the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the population of cells. In some such methods, nucleicacids encoding the chimeric Cas protein, the chimeric adaptor protein,the first tau repeat domain linked to the first reporter, and the secondtau repeat domain linked to the second reporter are genomicallyintegrated in the population of cells.

In some such methods, each guide RNA targets a guide RNA target sequencewithin 200 bp upstream of a transcription start site. In some suchmethods, each guide RNA comprises one or more adaptor-binding elementsto which the chimeric adaptor protein can specifically bind. Optionally,each guide RNA comprises two adaptor-binding elements to which thechimeric adaptor protein can specifically bind. Optionally, a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. Optionally, theadaptor-binding element comprises the sequence set forth in SEQ ID NO:33. Optionally, each of one or more guide RNAs is a single guide RNAcomprising a CRISPR RNA (crRNA) portion fused to a transactivatingCRISPR RNA (tracrRNA) portion, and the first loop is the tetraloopcorresponding to residues 13-16 of SEQ ID NO: 17, and the second loop isthe stem loop 2 corresponding to residues 53-56 of SEQ ID NO: 17.

In some such methods, step (c) is about 3 days to about 13 days. In somesuch methods, step (c) is about 7 days to about 10 days, is about 7days, or is about 10 days.

In some such methods, step (d) comprises culturing the geneticallymodified population of cells in the presence of a medium comprising acell lysate from cultured tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state. Optionally, thecell lysate in the medium is at a concentration of about 1 to about 5μg/mL. In some such methods, the medium comprising the cell lysatefurther comprises lipofectamine or another transfection reagent.Optionally, the medium comprising the cell lysate compriseslipofectamine at a concentration of about 1.5 to about 4 μL/mL. In somesuch methods, the genetically modified population of cells is notco-cultured with the tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state.

In some such methods, step (e) is about 1 day to about 3 days.Optionally, step (e) is about 2 days. In some such methods, the firstreporter and the second reporter are a fluorescence resonance energytransfer (FRET) pair, and the aggregation-positive population of cellsand the aggregation-negative population of cells in step (e) isidentified by flow cytometry. In some such methods, abundance isdetermined by next-generation sequencing.

In some such methods, a guide RNA is considered enriched in theaggregation-negative population of cells in step (e) if the abundance ofthe guide RNA relative to the total population of the plurality ofunique guide RNAs is at least 1.5-fold higher in theaggregation-negative population of cells in step (e) relative to theaggregation-positive population of cells in step (e) and/or the seededpopulation of cells in step (d), and wherein a guide RNA is considereddepleted in the aggregation-positive population of cells in step (e) ifthe abundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold lower in theaggregation-positive population of cells in step (e) relative to theaggregation-negative population of cells in step (e) and/or the seededpopulation of cells in step (d), or wherein a guide RNA is consideredenriched in the aggregation-positive population of cells in step (e) ifthe abundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to theaggregation-negative population of cells in step (e) and/or the seededpopulation of cells in step (d), and wherein a guide RNA is considereddepleted in the aggregation-negative population of cells in step (e) ifthe abundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold lower in theaggregation-negative population of cells in step (e) relative to theaggregation-positive population of cells in step (e) and/or the seededpopulation of cells in step (d).

In some such methods, step (f) comprises determining abundance of eachof the plurality of unique guide RNAs in the aggregation-negativepopulation of cells in step (e) relative to the aggregation-positivepopulation of cells in step (e), the cultured population of cells instep (c) at a first time point, and the seeded population of cells instep (d) at a second time point, and/or wherein step (f) comprisesdetermining abundance of each of the plurality of unique guide RNAs inthe aggregation-positive population of cells in step (e) relative to theaggregation-negative population of cells in step (e), the culturedpopulation of cells in step (c) at a first time point, and the seededpopulation of cells in step (d) at a second time point. Optionally, thefirst time point in step (c) is at a first passage of culturing thepopulation of cells. Optionally, the first time point in step (c) isafter about 3 days of culturing, and the second time point in step (c)is after about 7 days of culturing or about 10 days of culturing.

In some such methods, a gene is considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene prevents tau aggregation (or a candidate geneticmodifier of tau aggregation, wherein disruption or transcriptionalactivation of the gene is expected to prevent tau aggregation), if: (1)the abundance of a guide RNA targeting the gene relative to the totalpopulation of the plurality of unique guide RNAs is at least 1.5-foldhigher in the aggregation-negative population of cells in step (e)relative to the aggregation-positive population of cells in step (e),the cultured population of cells in step (c) at the first time point,and the seeded population of cells in step (d) at the second time point;and/or (2) the abundance of a guide RNA targeting the gene relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-negative population of cells in step(e) relative to the aggregation-positive population of cells in step (e)and the seeded population of cells in step (d) at the second time point;and/or (3) the abundance of a guide RNA targeting the gene relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold lower in the aggregation-positive population of cells in step(e) relative to the aggregation-negative population of cells in step(e), the cultured population of cells in step (c) at the first timepoint, and the seeded population of cells in step (d) at the second timepoint; and/or (4) the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-positive population ofcells in step (e) relative to the aggregation-negative population ofcells in step (e) and the seeded population of cells in step (d) at thesecond time point. In some such methods, a gene is considered a geneticmodifier of tau aggregation, wherein disruption (CRISPRn) ortranscriptional activation (CRISPRa) of the gene promotes or enhancestau aggregation (or a candidate genetic modifier of tau aggregation,wherein disruption or transcriptional activation of the gene is expectedto promote or enhance tau aggregation), if: (1) the abundance of a guideRNA targeting the gene relative to the total population of the pluralityof unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to theaggregation-negative population of cells in step (e), the culturedpopulation of cells in step (c) at the first time point, and the seededpopulation of cells in step (d) at the second time point; and/or (2) theabundance of a guide RNA targeting the gene relative to the totalpopulation of the plurality of unique guide RNAs is at least 1.5-foldhigher in the aggregation-positive population of cells in step (e)relative to the aggregation-negative population of cells in step (e) andthe seeded population of cells in step (d) at the second time point;and/or (3) the abundance of a guide RNA targeting the gene relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold lower in the aggregation-negative population of cells in step(e) relative to the aggregation-positive population of cells in step(e), the cultured population of cells in step (c) at the first timepoint, and the seeded population of cells in step (d) at the second timepoint; and/or (4) the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-negative population ofcells in step (e) relative to the aggregation-positive population ofcells in step (e) and the seeded population of cells in step (d) at thesecond time point.

In some such methods, the following steps are taken in step (f) toidentify a gene as a genetic modifier of tau aggregation, whereindisruption (CRISPRn) or transcriptional activation (CRISPRa) of the geneprevents tau aggregation (or as a candidate genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene is expected to prevent tau aggregation): (1)identifying which of the plurality of unique guide RNAs are present inthe aggregation-negative population of cells produced in step (e); (2)calculating the random chance of the guide RNAs identified in step(f)(1) being present using the formula nCn′*(x−n′)C(m−n)/xCm, wherein xis the variety of unique guide RNAs introduced into the population ofcells in step (b), wherein m is the variety of unique guide RNAsidentified in step (f)(1), wherein n is the variety of unique guide RNAsintroduced into the population of cells in step (b) that target thegene, and wherein n′ is the variety of unique guide RNAs identified instep (f)(1) that target the gene; (3) calculating average enrichmentscores for the guide RNAs identified in step (f)(1), wherein theenrichment score for a guide RNA is the relative abundance of the guideRNA in the aggregation-negative population of cells produced in step (e)divided by the relative abundance of the guide RNA in theaggregation-positive population of cells produced in step (e) or theseeded population of cells in step (d), and wherein relative abundanceis the read count of the guide RNA divided by the read count of thetotal population of the plurality of unique guide RNAs; and (4)selecting the gene if a guide RNA targeting the gene is significantlybelow the random chance of being present and above a thresholdenrichment score. In some such methods, the following steps are taken instep (f) to identify a gene as a genetic modifier of tau aggregation,wherein disruption (CRISPRn) or transcriptional activation (CRISPRa) ofthe gene promotes or enhances tau aggregation (or as a candidate geneticmodifier of tau aggregation, wherein disruption or transcriptionalactivation of the gene is expected to promote or enhance tauaggregation): (1) identifying which of the plurality of unique guideRNAs are present in the aggregation-positive population of cellsproduced in step (e); (2) calculating the random chance of the guideRNAs identified in step (f)(1) being present using the formulanCn′*(x−n′)C(m−n)/xCm, wherein x is the variety of unique guide RNAsintroduced into the population of cells in step (b), wherein m is thevariety of unique guide RNAs identified in step (f)(1), wherein n is thevariety of unique guide RNAs introduced into the population of cells instep (b) that target the gene, and wherein n′ is the variety of uniqueguide RNAs identified in step (f)(1) that target the gene; (3)calculating average enrichment scores for the guide RNAs identified instep (f)(1), wherein the enrichment score for a guide RNA is therelative abundance of the guide RNA in the aggregation-positivepopulation of cells produced in step (e) divided by the relativeabundance of the guide RNA in the aggregation-negative population ofcells produced in step (e) or the seeded population of cells in step(d), and wherein relative abundance is the read count of the guide RNAdivided by the read count of the total population of the plurality ofunique guide RNAs; and (4) selecting the gene if a guide RNA targetingthe gene is significantly below the random chance of being present andabove a threshold enrichment score.

In some such methods, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation.

In some such methods, the first tau repeat domain and/or the second taurepeat domain comprises a tau four-repeat domain. In some such methods,the first tau repeat domain and/or the second tau repeat domaincomprises SEQ ID NO: 11. In some such methods, the first tau repeatdomain and the second tau repeat domain are the same. In some suchmethods, the first tau repeat domain and the second tau repeat domainare the same and each comprises tau four-repeat domain comprising a tauP301S mutation.

In some such methods, first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such methods, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells.

In some such methods, the plurality of unique guide RNAs are introducedat a concentration selected such that a majority of the cells receiveonly one of the unique guide RNAs. In some such methods, the pluralityof unique guide RNAs target 100 or more genes, 1000 or more genes, or10000 or more genes. In some such methods, the library is a genome-widelibrary.

In some such methods, a plurality of target sequences are targeted onaverage in each of the targeted plurality of genes. Optionally, at leastthree target sequences are targeted on average in each of the targetedplurality of genes. Optionally, about three to about six targetsequences (e.g., about three, about four, or about six) are targeted onaverage in each of the targeted plurality of genes. Optionally, aboutthree target sequences are targeted on average in each of the targetedplurality of genes.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells by viral transduction. Optionally, each ofthe plurality of unique guide RNAs is in a separate viral vector.Optionally, the plurality of unique guide RNAs are introduced into thepopulation of cells by lentiviral transduction. In some such methods,the population of cells is infected at a multiplicity of infection ofless than about 0.3.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells together with a selection marker, and step(b) further comprises selecting cells that comprise the selectionmarker. Optionally, the selection marker imparts resistance to a drug.Optionally, the selection marker imparts resistance to puromycin orzeocin. Optionally, the selection marker is selected from neomycinphosphotransferase, hygromycin B phosphotransferase,puromycin-N-acetyltransferase, and blasticidin S deaminase. Optionally,the selection marker is selected from neomycin phosphotransferase,hygromycin B phosphotransferase, puromycin-N-acetyltransferase,blasticidin S deaminase, and bleomycin resistance protein.

In some such methods, the population of cells into which the pluralityof unique guide RNAs are introduced in step (b) comprises greater thanabout 300 cells per unique guide RNA.

In another aspect, provided are methods of screening for geneticmodifiers of tau aggregation and/or disaggregation. Some such methods(CRISPRn) comprise: (a) providing a population of cells comprising a Casprotein, a first tau repeat domain linked to a first reporter, and asecond tau repeat domain linked to a second reporter, wherein the cellsare tau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state; (b) introducing into the population ofcells a library comprising a plurality of unique guide RNAs that targeta plurality of genes; (c) culturing the population of cells to allowgenome editing and expansion, wherein the plurality of unique guide RNAsform complexes with the Cas protein, and the Cas protein cleaves theplurality of genes resulting in knockout of gene function to produce agenetically modified population of cells, and wherein the culturingresults in an aggregation-positive population of cells and anaggregation-negative population of cells; (d) identifying theaggregation-positive population of cells and the aggregation-negativepopulation of cells; and (e) determining abundance of each of theplurality of unique guide RNAs in the aggregation-positive population ofcells identified in step (d) relative to the aggregation-negativepopulation of cells identified in step (d) and/or the culturedpopulation of cells at one or more time points in step (c), and/ordetermining abundance of each of the plurality of unique guide RNAs inthe aggregation-negative population of cells identified in step (d)relative to the aggregation-positive population of cells identified instep (d) and/or the cultured population of cells at one or more timepoints in step (c), wherein enrichment of a guide RNA in theaggregation-negative population of cells identified in step (d) relativeto the aggregation-positive population of cells identified in step (d)and/or the cultured population of cells at one or more time points instep (c) or wherein depletion of a guide RNA in the aggregation-positivepopulation of cells identified in step (d) relative to theaggregation-negative population of cells identified in step (d) and/orcultured population of cells at one or more time points in step (c)indicates that the gene targeted by the guide RNA is a genetic modifierof tau disaggregation, wherein disruption of the gene targeted by theguide RNA promotes tau disaggregation, or is a candidate geneticmodifier of tau disaggregation (e.g., for further testing via secondaryscreens), wherein disruption of the gene targeted by the guide RNA isexpected to promote tau disaggregation, and/or wherein enrichment of aguide RNA in the aggregation-positive population of cells identified instep (d) relative to the aggregation-negative population of cellsidentified in step (d) and/or the cultured population of cells at one ormore time points in step (c) or wherein depletion of a guide RNA in theaggregation-negative population of cells identified in step (d) relativeto the aggregation-positive population of cells identified in step (d)and/or cultured population of cells at one or more time points in step(c) indicates that the gene targeted by the guide RNA is a geneticmodifier of tau aggregation, wherein disruption of the gene targeted bythe guide RNA promotes or enhances tau aggregation, or is a candidategenetic modifier of tau aggregation (e.g., for further testing viasecondary screens), wherein disruption of the gene targeted by the guideRNA is expected to promote or enhance tau aggregation.

In some such methods, the Cas protein is a Cas9 protein. Optionally, Casprotein is Streptococcus pyogenes Cas9. In some such methods, the Casprotein comprises SEQ ID NO: 21, optionally wherein the Cas protein isencoded by a coding sequence comprising the sequence set forth in SEQ IDNO: 22.

In some such methods, the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter are stably expressed in the population of cells. Insome such methods, nucleic acids encoding the Cas protein, the first taurepeat domain linked to the first reporter, and the second tau repeatdomain linked to the second reporter are genomically integrated in thepopulation of cells.

In some such methods, each guide RNA targets a constitutive exon.Optionally, each guide RNA targets a 5′ constitutive exon. In some suchmethods, each guide RNA targets a first exon, a second exon, or a thirdexon.

Some such methods (CRISPRa) comprise: (a) providing a population ofcells comprising a chimeric Cas protein comprising a nuclease-inactiveCas protein fused to one or more transcriptional activation domains, achimeric adaptor protein comprising an adaptor protein fused to one ormore transcriptional activation domains, a first tau repeat domainlinked to a first reporter, and a second tau repeat domain linked to asecond reporter, wherein the cells are tau-aggregation-positive cells inwhich a tau repeat domain stably presents in an aggregated state; (b)introducing into the population of cells a library comprising aplurality of unique guide RNAs that target a plurality of genes; (c)culturing the population of cells to allow transcriptional activationand expansion, wherein the plurality of unique guide RNAs form complexeswith the chimeric Cas protein and the chimeric adaptor protein, and thecomplexes activate transcription of the plurality of genes resulting inincreased gene expression to produce a genetically modified populationof cells, and wherein the culturing results in an aggregation-positivepopulation of cells and an aggregation-negative population of cells; (d)identifying the aggregation-positive population of cells and theaggregation-negative population of cells; and (e) determining abundanceof each of the plurality of unique guide RNAs in theaggregation-positive population of cells identified in step (d) relativeto the aggregation-negative population of cells identified in step (d)and/or the cultured population of cells at one or more time points instep (c), and/or determining abundance of each of the plurality ofunique guide RNAs in the aggregation-negative population of cellsidentified in step (d) relative to the aggregation-positive populationof cells identified in step (d) and/or the cultured population of cellsat one or more time points in step (c), wherein enrichment of a guideRNA in the aggregation-negative population of cells identified in step(d) relative to the aggregation-positive population of cells identifiedin step (d) and/or the cultured population of cells at one or more timepoints in step (c) or wherein depletion of a guide RNA in theaggregation-positive population of cells identified in step (d) relativeto the aggregation-negative population of cells identified in step (d)and/or cultured population of cells at one or more time points in step(c) indicates that the gene targeted by the guide RNA is a geneticmodifier of tau disaggregation, wherein transcriptional activation ofthe gene targeted by the guide RNA promotes tau disaggregation, or is acandidate genetic modifier of tau disaggregation (e.g., for furthertesting via secondary screens), wherein transcriptional activation ofthe gene targeted by the guide RNA is expected to promote taudisaggregation, and/or wherein enrichment of a guide RNA in theaggregation-positive population of cells identified in step (d) relativeto the aggregation-negative population of cells identified in step (d)and/or the cultured population of cells at one or more time points instep (c) or wherein depletion of a guide RNA in the aggregation-negativepopulation of cells identified in step (d) relative to theaggregation-positive population of cells identified in step (d) and/orcultured population of cells at one or more time points in step (c)indicates that the gene targeted by the guide RNA is a genetic modifierof tau aggregation, wherein transcriptional activation of the genetargeted by the guide RNA promotes or enhances tau aggregation, or is acandidate genetic modifier of tau aggregation (e.g., for further testingvia secondary screens), wherein transcriptional activation of the genetargeted by the guide RNA is expected to promote or enhance tauaggregation.

In some such methods, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. In some such methods, thechimeric Cas protein comprises the nuclease-inactive Cas protein fusedto a VP64 transcriptional activation domain, optionally wherein thechimeric Cas protein comprises from N-terminus to C-terminus: thenuclease-inactive Cas protein; a nuclear localization signal; and theVP64 transcriptional activator domain. In some such methods, the adaptorprotein is an MS2 coat protein, and wherein the one or moretranscriptional activation domains in the chimeric adaptor proteincomprise a p65 transcriptional activation domain and an HSF1transcriptional activation domain, optionally wherein the chimericadaptor protein comprises from N-terminus to C-terminus: the MS2 coatprotein; a nuclear localization signal; the p65 transcriptionalactivation domain; and the HSF1 transcriptional activation domain. Insome such methods, the chimeric Cas protein comprises SEQ ID NO: 36,optionally wherein the chimeric Cas protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 38. In somesuch methods, the chimeric adaptor protein comprises SEQ ID NO: 37,optionally wherein the chimeric adaptor protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 39.

In some such methods, the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the population of cells. In some such methods, nucleicacids encoding the chimeric Cas protein, the chimeric adaptor protein,the first tau repeat domain linked to the first reporter, and the secondtau repeat domain linked to the second reporter are genomicallyintegrated in the population of cells.

In some such methods, each guide RNA targets a guide RNA target sequencewithin 200 bp upstream of a transcription start site. In some suchmethods, each guide RNA comprises one or more adaptor-binding elementsto which the chimeric adaptor protein can specifically bind. Optionally,each guide RNA comprises two adaptor-binding elements to which thechimeric adaptor protein can specifically bind. Optionally, a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. Optionally, theadaptor-binding element comprises the sequence set forth in SEQ ID NO:33. Optionally, each of one or more guide RNAs is a single guide RNAcomprising a CRISPR RNA (crRNA) portion fused to a transactivatingCRISPR RNA (tracrRNA) portion, and the first loop is the tetraloopcorresponding to residues 13-16 of SEQ ID NO: 17, and the second loop isthe stem loop 2 corresponding to residues 53-56 of SEQ ID NO: 17.

In some such methods, step (c) is about 3 days to about 14 days.Optionally, step (c) is about 10 days to about 14 days or about 12 daysto about 14 days.

In some such methods, step (d) comprises synchronizing cell cycleprogression to obtain a cell population predominantly enriched in Sphase. Optionally, the synchronization is achieved by double thymidineblock.

In some such methods, the first reporter and the second reporter are afluorescence resonance energy transfer (FRET) pair, and theaggregation-positive population of cells and the aggregation-negativepopulation of cells in step (d) is identified by flow cytometry. In somesuch methods, abundance is determined by next-generation sequencing.

In some such methods, a guide RNA is considered enriched in theaggregation-negative population of cells in step (d) if the abundance ofthe guide RNA relative to the total population of the plurality ofunique guide RNAs is at least 1.5-fold higher in theaggregation-negative population of cells in step (d) relative to theaggregation-positive population of cells in step (d) and/or the culturedpopulation of cells at one or more time points in step (c), and whereina guide RNA is considered depleted in the aggregation-positivepopulation of cells in step (d) if the abundance of the guide RNArelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-positive population ofcells in step (d) relative to the aggregation-negative population ofcells in step (d) and/or the cultured population of cells at one or moretime points in step (c), or wherein a guide RNA is considered enrichedin the aggregation-positive population of cells in step (d) if theabundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (d) relative to theaggregation-negative population of cells in step (d) and/or the culturedpopulation of cells at one or more time points in step (c), and whereina guide RNA is considered depleted in the aggregation-negativepopulation of cells in step (d) if the abundance of the guide RNArelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-negative population ofcells in step (d) relative to the aggregation-positive population ofcells in step (d) and/or the cultured population of cells at one or moretime points in step (c).

In some such methods, step (e) comprises determining abundance of eachof the plurality of unique guide RNAs in the aggregation-negativepopulation of cells in step (d) relative to the aggregation-positivepopulation of cells in step (d), the cultured population of cells instep (c) at a first time point, and the cultured population of cells instep (c) at a second time point, and/or wherein step (e) comprisesdetermining abundance of each of the plurality of unique guide RNAs inthe aggregation-positive population of cells in step (d) relative to theaggregation-negative population of cells in step (e), the culturedpopulation of cells in step (c) at a first time point, and the culturedpopulation of cells in step (c) at a second time point. Optionally, thefirst time point in step (c) is at a first passage of culturing thepopulation of cells, and the second time point is in the middle ofculturing the population of cells to allow genome editing and expansionor transcriptional activation and expansion. Optionally, the first timepoint in step (c) is after about 7 days of culturing, and the secondtime point in step (c) is after about 10 days of culturing.

In some such methods, a gene is considered a genetic modifier of taudisaggregation, wherein disruption (CRISPRn) or transcriptionalactivation (CRISPRa) of the gene promotes tau disaggregation (or acandidate genetic modifier of tau disaggregation, wherein disruption ortranscriptional activation of the gene is expected to promote taudisaggregation), if: (1) the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold higher in the aggregation-negative population ofcells in step (d) relative to the aggregation-positive population ofcells in step (d), the cultured population of cells in step (c) at thefirst time point, and the cultured population of cells in step (c) atthe second time point; and/or (2) the abundance of a guide RNA targetingthe gene relative to the total population of the plurality of uniqueguide RNAs is at least 1.5-fold higher in the aggregation-negativepopulation of cells in step (d) relative to the aggregation-positivepopulation of cells in step (d) and the cultured population of cells instep (c) at the second time point; and/or (3) the abundance of a guideRNA targeting the gene relative to the total population of the pluralityof unique guide RNAs is at least 1.5-fold lower in theaggregation-positive population of cells in step (d) relative to theaggregation-negative population of cells in step (d), the culturedpopulation of cells in step (c) at the first time point, and thecultured population of cells in step (c) at the second time point;and/or (4) the abundance of a guide RNA targeting the gene relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold lower in the aggregation-positive population of cells in step(d) relative to the aggregation-negative population of cells in step (d)and the cultured population of cells in step (c) at the second timepoint. In some such methods, a gene is considered a genetic modifier oftau aggregation, wherein disruption (CRISPRn) or transcriptionalactivation (CRISPRa) of the gene promotes or enhances tau aggregation(or a candidate genetic modifier of tau aggregation, wherein disruptionor transcriptional activation of the gene is expected to promote orenhance tau aggregation), if: (1) the abundance of a guide RNA targetingthe gene relative to the total population of the plurality of uniqueguide RNAs is at least 1.5-fold higher in the aggregation-positivepopulation of cells in step (d) relative to the aggregation-negativepopulation of cells in step (d), the cultured population of cells instep (c) at the first time point, and the cultured population of cellsin step (c) at the second time point; and/or (2) the abundance of aguide RNA targeting the gene relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (d) relative to theaggregation-negative population of cells in step (d) and the culturedpopulation of cells in step (c) at the second time point; and/or (3) theabundance of a guide RNA targeting the gene relative to the totalpopulation of the plurality of unique guide RNAs is at least 1.5-foldlower in the aggregation-negative population of cells in step (d)relative to the aggregation-positive population of cells in step (d),the cultured population of cells in step (c) at the first time point,and the cultured population of cells in step (c) at the second timepoint; and/or (4) the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-negative population ofcells in step (d) relative to the aggregation-positive population ofcells in step (d) and the cultured population of cells in step (c) atthe second time point.

In some such methods, the following steps are taken in step (e) toidentify a gene as a genetic modifier of tau disaggregation, whereindisruption (CRISPRn) or transcriptional activation (CRISPRa) of the genepromotes tau disaggregation (or a candidate genetic modifier of taudisaggregation, wherein disruption or transcriptional activation of thegene is expected to promote tau disaggregation): (1) identifying whichof the plurality of unique guide RNAs are present in theaggregation-negative population of cells identified in step (d); (2)calculating the random chance of the guide RNAs identified in step(e)(1) being present using the formula nCn′*(x−n′)C(m−n)/xCm, wherein xis the variety of unique guide RNAs introduced into the population ofcells in step (b), wherein m is the variety of unique guide RNAsidentified in step (e)(1), wherein n is the variety of unique guide RNAsintroduced into the population of cells in step (b) that target thegene, and wherein n′ is the variety of unique guide RNAs identified instep (e)(1) that target the gene; (3) calculating average enrichmentscores for the guide RNAs identified in step (e)(1), wherein theenrichment score for a guide RNA is the relative abundance of the guideRNA in the aggregation-negative population of cells identified in step(d) divided by the relative abundance of the guide RNA in theaggregation-positive population of cells identified in step (d) or thecultured population of cells in step (c) at the first time point or thesecond time point, and wherein relative abundance is the read count ofthe guide RNA divided by the read count of the total population of theplurality of unique guide RNAs; and (4) selecting the gene if a guideRNA targeting the gene is significantly below the random chance of beingpresent and above a threshold enrichment score. In some such methods,the following steps are taken in step (e) to identify a gene as agenetic modifier of tau aggregation, wherein disruption (CRISPRn) ortranscriptional activation (CRISPRa) of the gene promotes or enhancestau aggregation (or a candidate genetic modifier of tau aggregation,wherein disruption or transcriptional activation of the gene is expectedto promote or enhance tau aggregation): (1) identifying which of theplurality of unique guide RNAs are present in the aggregation-positivepopulation of cells identified in step (d); (2) calculating the randomchance of the guide RNAs identified in step (e)(1) being present usingthe formula nCn′*(x−n′)C(m−n)/xCm, wherein x is the variety of uniqueguide RNAs introduced into the population of cells in step (b), whereinm is the variety of unique guide RNAs identified in step (e)(1), whereinn is the variety of unique guide RNAs introduced into the population ofcells in step (b) that target the gene, and wherein n′ is the variety ofunique guide RNAs identified in step (e)(1) that target the gene; (3)calculating average enrichment scores for the guide RNAs identified instep (e)(1), wherein the enrichment score for a guide RNA is therelative abundance of the guide RNA in the aggregation-positivepopulation of cells identified in step (d) divided by the relativeabundance of the guide RNA in the aggregation-negative population ofcells identified in step (d) or the cultured population of cells in step(c) at the first time point or the second time point, and whereinrelative abundance is the read count of the guide RNA divided by theread count of the total population of the plurality of unique guideRNAs; and (4) selecting the gene if a guide RNA targeting the gene issignificantly below the random chance of being present and above athreshold enrichment score.

In some such methods, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such methods, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation.

In some such methods, the first tau repeat domain and/or the second taurepeat domain comprises a tau four-repeat domain. In some such methods,the first tau repeat domain and/or the second tau repeat domaincomprises SEQ ID NO: 11. In some such methods, the first tau repeatdomain and the second tau repeat domain are the same. In some suchmethods, the first tau repeat domain and the second tau repeat domainare the same and each comprises tau four-repeat domain comprising a tauP301S mutation.

In some such methods, first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such methods, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells.

In some such methods, the plurality of unique guide RNAs are introducedat a concentration selected such that a majority of the cells receiveonly one of the unique guide RNAs. In some such methods, the pluralityof unique guide RNAs target 100 or more genes, 1000 or more genes, or10000 or more genes. In some such methods, the library is a genome-widelibrary.

In some such methods, a plurality of target sequences are targeted onaverage in each of the targeted plurality of genes. Optionally, at leastthree target sequences are targeted on average in each of the targetedplurality of genes. Optionally, about three to about six targetsequences (e.g., about three, about four, or about six) are targeted onaverage in each of the targeted plurality of genes. Optionally, aboutthree target sequences are targeted on average in each of the targetedplurality of genes.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells by viral transduction. Optionally, each ofthe plurality of unique guide RNAs is in a separate viral vector.Optionally, the plurality of unique guide RNAs are introduced into thepopulation of cells by lentiviral transduction. In some such methods,the population of cells is infected at a multiplicity of infection ofless than about 0.3.

In some such methods, the plurality of unique guide RNAs are introducedinto the population of cells together with a selection marker, and step(b) further comprises selecting cells that comprise the selectionmarker. Optionally, the selection marker imparts resistance to a drug.Optionally, the selection marker imparts resistance to puromycin orzeocin. Optionally, the selection marker is selected from neomycinphosphotransferase, hygromycin B phosphotransferase,puromycin-N-acetyltransferase, and blasticidin S deaminase. Optionally,the selection marker is selected from neomycin phosphotransferase,hygromycin B phosphotransferase, puromycin-N-acetyltransferase,blasticidin S deaminase, and bleomycin resistance protein.

In some such methods, the population of cells into which the pluralityof unique guide RNAs are introduced in step (b) comprises greater thanabout 300 cells per unique guide RNA.

In another aspect, provided are Cas-tau biosensor cells or populationsof such cells. Some such cells comprise a population of one or morecells comprising a Cas protein, a first tau repeat domain linked to afirst reporter, and a second tau repeat domain linked to a secondreporter.

In some such cells, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such cells, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation.

In some such cells, the first tau repeat domain and/or the second taurepeat domain comprises a tau four-repeat domain. In some such cells,the first tau repeat domain and/or the second tau repeat domaincomprises SEQ ID NO: 11. In some such cells, the first tau repeat domainand the second tau repeat domain are the same. In some such cells, thefirst tau repeat domain and the second tau repeat domain are the sameand each comprises tau four-repeat domain comprising a tau P301Smutation.

In some such cells, the first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such cells, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. Optionally, the Cas proteincomprises SEQ ID NO: 21. Optionally, the Cas protein is encoded by acoding sequence comprising the sequence set forth in SEQ ID NO: 22.

In some such cells, the Cas protein, the first tau repeat domain linkedto the first reporter, and the second tau repeat domain linked to thesecond reporter are stably expressed in the cell. In some such cells,nucleic acids encoding the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter are genomically integrated in the cell.

In some such cells, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells. Some such cells are in vitro.

In some such cells, wherein the first tau repeat domain linked to thefirst reporter and the second tau repeat domain linked to the secondreporter are not stably present in an aggregated state. In some suchcells, the first tau repeat domain linked to the first reporter and thesecond tau repeat domain linked to the second reporter stably present inan aggregated state.

In another aspect, provided are in vitro cultures of Cas-tau biosensorcells and conditioned medium. Some such in vitro cultures comprise anyof the populations of cells described above or elsewhere herein and aculture medium comprising a conditioned medium harvested from culturedtau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state.

In some such in vitro cultures, the conditioned medium was harvestedafter being on confluent tau-aggregation-positive cells for about 1 toabout 7 days. Optionally, the conditioned medium was harvested afterbeing on confluent tau-aggregation-positive cells for about 4 days.

In some such in vitro cultures, the culture medium comprises about 75%conditioned medium and about 25% fresh medium. In some such in vitrocultures, the population of cells is not co-cultured with the culturedtau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state.

In another aspect, provided are SAM-tau biosensor cells or populationsof such cells. Some such cells comprise a population of one or morecells comprising a chimeric Cas protein comprising a nuclease-inactiveCas protein fused to one or more transcriptional activation domains, achimeric adaptor protein comprising an adaptor protein fused to one ormore transcriptional activation domains, a first tau repeat domainlinked to a first reporter, and a second tau repeat domain linked to asecond reporter.

In some such cells, the first tau repeat domain and/or the second taurepeat domain is a human tau repeat domain. In some such cells, thefirst tau repeat domain and/or the second tau repeat domain comprises apro-aggregation mutation. Optionally, the first tau repeat domain and/orthe second tau repeat domain comprises a tau P301S mutation.

In some such cells, the first tau repeat domain and/or the second taurepeat domain comprises a tau four-repeat domain. In some such cells,the first tau repeat domain and/or the second tau repeat domaincomprises SEQ ID NO: 11. In some such cells, the first tau repeat domainand the second tau repeat domain are the same. In some such cells, thefirst tau repeat domain and the second tau repeat domain are the sameand each comprises tau four-repeat domain comprising a tau P301Smutation.

In some such cells, the first reporter and the second reporter arefluorescent proteins. Optionally, the first reporter and the secondreporter are a fluorescence resonance energy transfer (FRET) pair.Optionally, the first reporter is cyan fluorescent protein (CFP) and thesecond reporter is yellow fluorescent protein (YFP).

In some such cells, the Cas protein is a Cas9 protein. Optionally, theCas protein is Streptococcus pyogenes Cas9. In some such cells, thechimeric Cas protein comprises the nuclease-inactive Cas protein fusedto a VP64 transcriptional activation domain, optionally wherein thechimeric Cas protein comprises from N-terminus to C-terminus: thenuclease-inactive Cas protein; a nuclear localization signal; and theVP64 transcriptional activator domain. In some such cells, the adaptorprotein is an MS2 coat protein, and wherein the one or moretranscriptional activation domains in the chimeric adaptor proteincomprise a p65 transcriptional activation domain and an HSF1transcriptional activation domain, optionally wherein the chimericadaptor protein comprises from N-terminus to C-terminus: the MS2 coatprotein; a nuclear localization signal; the p65 transcriptionalactivation domain; and the HSF1 transcriptional activation domain. Insome such cells, the chimeric Cas protein comprises SEQ ID NO: 36,optionally wherein the chimeric Cas protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 38. In somesuch cells, the chimeric adaptor protein comprises SEQ ID NO: 37,optionally wherein the chimeric adaptor protein is encoded by a codingsequence comprising the sequence set forth in SEQ ID NO: 39.

In some such cells, the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the cell. In some such cells, nucleic acids encoding thechimeric Cas protein, the chimeric adaptor protein, the first tau repeatdomain linked to the first reporter, and the second tau repeat domainlinked to the second reporter are genomically integrated in the cell.

In some such cells, the cells are eukaryotic cells. Optionally, thecells are mammalian cells. Optionally, the cells are human cells.Optionally, the cells are HEK293T cells. Some such cells are in vitro.

In some such cells, wherein the first tau repeat domain linked to thefirst reporter and the second tau repeat domain linked to the secondreporter are not stably present in an aggregated state. In some suchcells, the first tau repeat domain linked to the first reporter and thesecond tau repeat domain linked to the second reporter stably present inan aggregated state.

In another aspect, provided are in vitro cultures of SAM-tau biosensorcells and conditioned medium. Some such in vitro cultures comprise anyof the populations of cells described above or elsewhere herein and aculture medium comprising a conditioned medium harvested from culturedtau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state.

In some such in vitro cultures, the conditioned medium was harvestedafter being on confluent tau-aggregation-positive cells for about 1 toabout 7 days. Optionally, the conditioned medium was harvested afterbeing on confluent tau-aggregation-positive cells for about 4 days.

In some such in vitro cultures, the culture medium comprises about 75%conditioned medium and about 25% fresh medium. In some such in vitrocultures, the population of cells is not co-cultured with the culturedtau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state.

In another aspect, provided are in vitro cultures of Cas-tau biosensorcells or SAM-tau biosensor cells and a culture medium comprising a celllysate from cultured tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state. Some such in vitrocultures comprise any of the populations of cells described above orelsewhere herein.

In some such in vitro cultures, the cell lysate in the medium is at aconcentration of about 1 to about 5 μg/mL. In some such in vitrocultures, the medium comprising the cell lysate further compriseslipofectamine or another transfection reagent. Optionally, the mediumcomprising the cell lysate comprises lipofectamine at a concentration ofabout 1.5 to about 4 L/mL. In some such in vitro cultures, the celllysate was produced by sonication of the tau-aggregation-positive cellsfor about 2 minutes to about 4 minutes after collecting the cells in abuffer comprising protease inhibitors.

In another aspect, provided are methods of producing conditioned mediumfor inducing or sensitizing to tau aggregation. Some such methodscomprise: (a) providing a population of tau-aggregation-positive cellsin which a tau repeat domain stably presents in an aggregated state; (b)culturing the population of tau-aggregation-positive cells in a mediumto produce a conditioned medium; and (c) harvesting the conditionedmedium.

In some such methods, the tau-aggregation-positive cells are cultured instep (b) to confluence. Optionally, the conditioned medium is harvestedafter being on the confluent tau-aggregation-positive cells in step (c)for about 1 to about 7 days. Optionally, the conditioned medium isharvested after being on the confluent tau-aggregation-positive cells instep (c) for about 4 days.

In another aspect, provided are methods of generating a population oftau-aggregation-positive cells. Some such methods comprise: (a)producing a conditioned medium for inducing tau aggregation according toany of the methods described above or elsewhere herein; and (b)culturing a population of cells comprising a protein comprising a taurepeat domain in a culture medium comprising the conditioned medium toproduce the population of tau-aggregation-positive cells.

In some such methods, the culture medium comprises about 75% conditionedmedium and about 25% fresh medium. In some such methods, the populationof cells is not co-cultured with the tau-aggregation-positive cells usedin the method to produce the conditioned medium.

In some such methods, the tau repeat domain comprises a pro-aggregationmutation. In some such methods, the tau repeat domain comprises a tauP301S mutation. In some such methods, the tau repeat domain comprises atau four-repeat domain. In some such methods, the tau repeat domaincomprises SEQ ID NO: 11.

In another aspect, provided are methods of producing a medium comprisinga cell lysate from cultured tau-aggregation-positive cells for inducingtau aggregation. Some such methods comprise: (a) providing a populationof tau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state; (b) collecting thetau-aggregation-positive cells in a buffer comprising proteaseinhibitors; (c) sonicating the tau-aggregation-positive cells for about2 minutes to about 4 minutes to produce the cell lysate; and (d) addingthe cell lysate to a growth medium.

In some such methods, the cell lysate in the growth medium is at aconcentration of about 1 to about 5 μg/mL. Some such methods furthercomprise adding lipofectamine or another transfection reagent to thegrowth medium in step (d). Optionally, step (d) comprising addinglipofectamine at a concentration of about 1.5 to about 4 μL/mL.

In another aspect, provided are methods of generating a population oftau-aggregation-positive cells. Some such methods comprise: (a)producing a medium comprising a cell lysate from culturedtau-aggregation-positive cells according to the any of the abovemethods; and (b) culturing a population of cells comprising a proteincomprising a tau repeat domain in the medium comprising a cell lysatefrom cultured tau-aggregation-positive cells.

In some such methods, the population of cells is not co-cultured withthe tau-aggregation-positive cells used in the method to produce theconditioned medium.

In some such methods, the tau repeat domain comprises a pro-aggregationmutation. In some such methods, the tau repeat domain comprises a tauP301S mutation. In some such methods, the tau repeat domain comprises atau four-repeat domain. In some such methods, the tau repeat domaincomprises SEQ ID NO: 11.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (not to scale) shows a schematic of tau isoform 2N4R. The taubiosensor lines include only tau4RD-YFP and tau4RD-CFP as transgenes,not the full 2N4R.

FIG. 2 shows a schematic of how aggregate formation is monitored byfluorescence resonance energy transfer (FRET) in tau biosensor celllines. The tau^(4RD)-CFP protein is excited by violet light and emitsblue light. The tau^(4RD)-YFP fusion protein is excited by blue lightand emits yellow light. If there is no aggregation, excitation by violetlight will not lead to FRET. If there is tau aggregation, excitation byviolet light will lead to FRET and yellow light emission.

FIG. 3A shows relative Cas9 mRNA expression intau^(4RD)-CFP/tau^(4RD)-YFP (TCY) biosensor cell clones transduced withlentiviral Cas9 expression constructs relative to clone Cas9H1, which isa control underperforming Cas9-expression TCY clone that was previouslyisolated.

FIG. 3B shows cutting efficiency at the PERK locus and the SNCA locus inthe Cas9 TCY clones three and seven days after transduction with sgRNAstargeting PERK and SNCA respectively.

FIG. 4 shows a schematic of the strategy for disruption of target genesin Cas9 TCY biosensor cell using a genome-wide CRISPR/Cas9 sgRNAlibrary.

FIG. 5 is a schematic showing derivation of tau^(4RD)-YFP Agg[+]subclones containing stably propagating tau aggregates whentau^(4RD)-YFP cells are seeded with tau^(4RD) fibrils. A fluorescencemicroscopy image showing the subclone with tau aggregates is also shown.

FIG. 6 is a schematic showing that conditioned medium from tau^(4RD)-YFPAgg[+] subclones collected after three days on confluent cells canprovide a source of tau aggregation activity whereas medium fromtau^(4RD)-YFP Agg[−] subclones does not. Conditioned medium was appliedto recipient cells as 75% conditioned medium and 25% fresh medium.Fluorescence-activated cell sorting (FACS) analysis images are shown foreach. The x-axis shows CFP (405 nm laser excitation), and the y-axisshows FRET (excitation from CFP emission). The upper right quadrant isFRET[+], the lower right quadrant is CFP[+], and the lower left quadrantis double-negative.

FIG. 7 is a schematic showing the strategy for a genome-wide CRISPRnuclease (CRISPRn) screen to identify modifier genes that promote tauaggregation.

FIG. 8 is a schematic showing the concepts of abundance and enrichmentfor next-generation sequencing (NGS) analysis using the gnome-wideCRISPRn screen.

FIG. 9 shows a schematic for secondary screening for Target Genes 1-14identified in the genome-wide screen for modifier genes that promote tauaggregation.

FIG. 10 is a graph showing FRET induction by tau aggregate conditionedmedium in Cas9 TCY biosensor cells transduced with lentiviral expressionconstructs for sgRNAs targeting Target Genes 1-14. The secondary screenconfirmed that Target Genes 2 and 8 modulate cell susceptibility to tauseeding/aggregation.

FIG. 11 shows FACS analysis images for Cas9 TCY biosensor cellstransduced with lentiviral expression constructs for Target Gene 2gRNA1, Target Gene 8 gRNA5, a non-targeting gRNA, and no gRNA. The cellswere cultured in conditioned medium or fresh medium. The x-axis showsCFP (405 nm laser excitation), and the y-axis shows FRET (excitationfrom CFP emission). The upper right quadrant is FRET[+], the lower rightquadrant is CFP[+], and the lower left quadrant is double-negative.Disruption of Target Gene 2 or 8 increases the formation of tauaggregates in response to tau aggregate conditioned medium but not freshmedium.

FIG. 12 shows a schematic for secondary screening in Cas9 TCY biosensorcells transduced with lentiviral expression constructs for sgRNAstargeting Target Genes 2 and 8, including mRNA expression analysis,protein expression analysis, and FRET analysis. Two sgRNAs were usedagainst Target Gene 2 (g1 and g3), one sgRNA was used against TargetGene 8 (g5), and a non-targeting sgRNA (g3) was used as a non-targetingcontrol.

FIG. 13 shows relative expression of Target Gene 2 and Target Gene 8 inCas9 TCY biosensor cells as assessed by qRT-PCR at Day 6 followingtransduction with the lentiviral sgRNA expression constructs.

FIG. 14 shows expression of Protein 2 (encoded by Target Gene 2) andProtein 8 (encoded by Target Gene 8) in Cas9 TCY biosensor cells asassessed by western blot at Day 13 following transduction with thelentiviral sgRNA expression constructs.

FIG. 15 shows tau aggregation as measured by percent FRET[+] cells inCas9 TCY biosensor cells at Day 10 following transduction with thelentiviral sgRNA expression constructs. No lipofectamine was used.

FIG. 16 shows expression of Target Gene 2 and Target Gene 8 in theknockdown Cas9 TCY cell clones as assessed by western blot.

FIG. 17 shows expression of tau aggregation in the Target Gene 2 andTarget Gene 8 knockdown Cas9 TCY cell clones as assessed by FRET.

FIG. 18 shows expression of Target Gene 2 and Target Gene 8 in theknockdown Cas9 TCY cell clones as assessed by western blot andphosphorylation of tau at positions S262 and S356 in those clones asassessed by western blot.

FIG. 19 shows whole cell lysate from tau-YFP Agg[+] clone18 can inducetau aggregation and FRET signal in tau biosensor cells. Differentamounts of whole cell lysate were tested, and different sonicationconditions for producing the lysate were tested.

FIG. 20 shows whole cell lysate from tau-YFP Agg[+] clone18 can inducetau aggregation and FRET signal in tau biosensor cells. Differentamounts of whole cell lysate were tested, and different amounts oflipofectamine were tested.

FIG. 21 shows whole cell lysate from tau-YFP Agg[+] clone18 can inducetau aggregation and FRET signal in tau biosensor cells but whole celllysate from Agg[−] clones cannot. Different amounts of whole cell lysatewere tested, and different amounts of lipofectamine were tested.

FIG. 22 shows a schematic showing the strategy for a genome-wide CRISPRnuclease (CRISPRn) screen to identify modifier genes that prevent tauaggregation.

FIG. 23 is a graph showing the identification of genes with uniquelyenriched sgRNAs in FRET[−] samples.

FIG. 24 is a graph showing the identification of genes with uniquelydepleted sgRNAs in FRET[−] samples.

FIG. 25 shows a schematic showing the strategy for secondary screeningto confirm identified modifier genes that prevent tau aggregation.

FIG. 26 shows a schematic showing the strategy for a genome-wide CRISPRactivation (CRISPRa) screen to identify modifier genes that prevent tauaggregation.

FIG. 27 shows a schematic showing the strategy for a genome-wide CRISPRnuclease (CRISPRn) screen to identify modifier genes that promote taudisaggregation.

FIG. 28 shows gating used for sorting Agg[+], speckles[+], and Agg[−]cell populations.

FIG. 29 shows a schematic for a thymidine block strategy used in thegenome-wide CRISPR nuclease (CRISPRn) screen to identify modifier genesthat promote tau disaggregation.

DEFINITIONS

The terms “protein,” “polypeptide,” and “peptide,” used interchangeablyherein, include polymeric forms of amino acids of any length, includingcoded and non-coded amino acids and chemically or biochemically modifiedor derivatized amino acids. The terms also include polymers that havebeen modified, such as polypeptides having modified peptide backbones.The term “domain” refers to any part of a protein or polypeptide havinga particular function or structure.

Proteins are said to have an “N-terminus” and a “C-terminus.” The term“N-terminus” relates to the start of a protein or polypeptide,terminated by an amino acid with a free amine group (—NH2). The term“C-terminus” relates to the end of an amino acid chain (protein orpolypeptide), terminated by a free carboxyl group (—COOH).

The terms “nucleic acid” and “polynucleotide,” used interchangeablyherein, include polymeric forms of nucleotides of any length, includingribonucleotides, deoxyribonucleotides, or analogs or modified versionsthereof. They include single-, double-, and multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purinebases, pyrimidine bases, or other natural, chemically modified,biochemically modified, non-natural, or derivatized nucleotide bases.

Nucleic acids are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. An end of an oligonucleotide is referred to as the “5′ end” ifits 5′ phosphate is not linked to the 3′ oxygen of a mononucleotidepentose ring. An end of an oligonucleotide is referred to as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of anothermononucleotide pentose ring. A nucleic acid sequence, even if internalto a larger oligonucleotide, also may be said to have 5′ and 3′ ends. Ineither a linear or circular DNA molecule, discrete elements are referredto as being “upstream” or 5′ of the “downstream” or 3′ elements.

The term “genomically integrated” refers to a nucleic acid that has beenintroduced into a cell such that the nucleotide sequence integrates intothe genome of the cell. Any protocol may be used for the stableincorporation of a nucleic acid into the genome of a cell.

The term “targeting vector” refers to a recombinant nucleic acid thatcan be introduced by homologous recombination,non-homologous-end-joining-mediated ligation, or any other means ofrecombination to a target position in the genome of a cell.

The term “viral vector” refers to a recombinant nucleic acid thatincludes at least one element of viral origin and includes elementssufficient for or permissive of packaging into a viral vector particle.The vector and/or particle can be utilized for the purpose oftransferring DNA, RNA, or other nucleic acids into cells either ex vivoor in vivo. Numerous forms of viral vectors are known.

The term “wild type” includes entities having a structure and/oractivity as found in a normal (as contrasted with mutant, diseased,altered, or so forth) state or context. Wild type genes and polypeptidesoften exist in multiple different forms (e.g., alleles).

The term “endogenous sequence” refers to a nucleic acid sequence thatoccurs naturally within a cell or organism. For example, an endogenousMAPT sequence of a cell or organism refers to a native MAPT sequencethat naturally occurs at the MAPT locus in the cell or organism.

“Exogenous” molecules or sequences include molecules or sequences thatare not normally present in a cell in that form. Normal presenceincludes presence with respect to the particular developmental stage andenvironmental conditions of the cell. An exogenous molecule or sequence,for example, can include a mutated version of a corresponding endogenoussequence within the cell, such as a humanized version of the endogenoussequence, or can include a sequence corresponding to an endogenoussequence within the cell but in a different form (i.e., not within achromosome). In contrast, endogenous molecules or sequences includemolecules or sequences that are normally present in that form in aparticular cell at a particular developmental stage under particularenvironmental conditions.

The term “heterologous” when used in the context of a nucleic acid or aprotein indicates that the nucleic acid or protein comprises at leasttwo segments that do not naturally occur together in the same molecule.For example, the term “heterologous,” when used with reference tosegments of a nucleic acid or segments of a protein, indicates that thenucleic acid or protein comprises two or more sub-sequences that are notfound in the same relationship to each other (e.g., joined together) innature. As one example, a “heterologous” region of a nucleic acid vectoris a segment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a nucleic acid vectorcould include a coding sequence flanked by sequences not found inassociation with the coding sequence in nature. Likewise, a“heterologous” region of a protein is a segment of amino acids within orattached to another peptide molecule that is not found in associationwith the other peptide molecule in nature (e.g., a fusion protein, or aprotein with a tag). Similarly, a nucleic acid or protein can comprise aheterologous label or a heterologous secretion or localization sequence.

The term “locus” refers to a specific location of a gene (or significantsequence), DNA sequence, polypeptide-encoding sequence, or position on achromosome of the genome of an organism. For example, a “MAPT locus” mayrefer to the specific location of a MAPT gene, MAPT DNA sequence,microtubule-associated-protein-tau-encoding sequence, or MAPT positionon a chromosome of the genome of an organism that has been identified asto where such a sequence resides. A “MAPT locus” may comprise aregulatory element of a MAPT gene, including, for example, an enhancer,a promoter, 5′ and/or 3′ untranslated region (UTR), or a combinationthereof.

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (e.g., an RNA product and/or a polypeptide product) andincludes the coding region interrupted with non-coding introns andsequence located adjacent to the coding region on both the 5′ and 3′ends such that the gene corresponds to the full-length mRNA (includingthe 5′ and 3′ untranslated sequences). The term “gene” also includesother non-coding sequences including regulatory sequences (e.g.,promoters, enhancers, and transcription factor binding sites),polyadenylation signals, internal ribosome entry sites, silencers,insulating sequence, and matrix attachment regions. These sequences maybe close to the coding region of the gene (e.g., within 10 kb) or atdistant sites, and they influence the level or rate of transcription andtranslation of the gene.

The term “allele” refers to a variant form of a gene. Some genes have avariety of different forms, which are located at the same position, orgenetic locus, on a chromosome. A diploid organism has two alleles ateach genetic locus. Each pair of alleles represents the genotype of aspecific genetic locus. Genotypes are described as homozygous if thereare two identical alleles at a particular locus and as heterozygous ifthe two alleles differ.

A “promoter” is a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particularpolynucleotide sequence. A promoter may additionally comprise otherregions which influence the transcription initiation rate. The promotersequences disclosed herein modulate transcription of an operably linkedpolynucleotide. A promoter can be active in one or more of the celltypes disclosed herein (e.g., a human cell, a pluripotent cell, aone-cell stage embryo, a differentiated cell, or a combination thereof).A promoter can be, for example, a constitutively active promoter, aconditional promoter, an inducible promoter, a temporally restrictedpromoter (e.g., a developmentally regulated promoter), or a spatiallyrestricted promoter (e.g., a cell-specific or tissue-specific promoter).Examples of promoters can be found, for example, in WO 2013/176772,herein incorporated by reference in its entirety for all purposes.

“Operable linkage” or being “operably linked” includes juxtaposition oftwo or more components (e.g., a promoter and another sequence element)such that both components function normally and allow the possibilitythat at least one of the components can mediate a function that isexerted upon at least one of the other components. For example, apromoter can be operably linked to a coding sequence if the promotercontrols the level of transcription of the coding sequence in responseto the presence or absence of one or more transcriptional regulatoryfactors. Operable linkage can include such sequences being contiguouswith each other or acting in trans (e.g., a regulatory sequence can actat a distance to control transcription of the coding sequence).

The term “variant” refers to a nucleotide sequence differing from thesequence most prevalent in a population (e.g., by one nucleotide) or aprotein sequence different from the sequence most prevalent in apopulation (e.g., by one amino acid).

The term “fragment” when referring to a protein means a protein that isshorter or has fewer amino acids than the full-length protein. The term“fragment” when referring to a nucleic acid means a nucleic acid that isshorter or has fewer nucleotides than the full-length nucleic acid. Afragment can be, for example, an N-terminal fragment (i.e., removal of aportion of the C-terminal end of the protein), a C-terminal fragment(i.e., removal of a portion of the N-terminal end of the protein), or aninternal fragment.

“Sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences refers to the residues in the two sequencesthat are the same when aligned for maximum correspondence over aspecified comparison window. When percentage of sequence identity isused in reference to proteins, residue positions which are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known. Typically, this involves scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, California).

“Percentage of sequence identity” includes the value determined bycomparing two optimally aligned sequences (greatest number of perfectlymatched residues) over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Unless otherwise specified(e.g., the shorter sequence includes a linked heterologous sequence),the comparison window is the full length of the shorter of the twosequences being compared.

Unless otherwise stated, sequence identity/similarity values include thevalue obtained using GAP Version 10 using the following parameters: %identity and % similarity for a nucleotide sequence using GAP Weight of50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using GAP Weight of8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or anyequivalent program thereof. “Equivalent program” includes any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The term “conservative amino acid substitution” refers to thesubstitution of an amino acid that is normally present in the sequencewith a different amino acid of similar size, charge, or polarity.Examples of conservative substitutions include the substitution of anon-polar (hydrophobic) residue such as isoleucine, valine, or leucinefor another non-polar residue. Likewise, examples of conservativesubstitutions include the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, or between glycine and serine. Additionally,the substitution of a basic residue such as lysine, arginine, orhistidine for another, or the substitution of one acidic residue such asaspartic acid or glutamic acid for another acidic residue are additionalexamples of conservative substitutions. Examples of non-conservativesubstitutions include the substitution of a non-polar (hydrophobic)amino acid residue such as isoleucine, valine, leucine, alanine, ormethionine for a polar (hydrophilic) residue such as cysteine,glutamine, glutamic acid or lysine and/or a polar residue for anon-polar residue. Typical amino acid categorizations are summarizedbelow.

TABLE 1 Amino Acid Categorizations. Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive −4.5 Asparagine Asn N Polar Neutral −3.5Aspartic acid Asp D Polar Negative −3.5 Cysteine Cys C Nonpolar Neutral2.5 Glutamic acid Glu E Polar Negative −3.5 Glutamine Gln Q PolarNeutral −3.5 Glycine Gly G Nonpolar Neutral −0.4 Histidine His H PolarPositive −3.2 Isoleucine Ile I Nonpolar Neutral 4.5 Leucine Leu LNonpolar Neutral 3.8 Lysine Lys K Polar Positive −3.9 Methionine Met MNonpolar Neutral 1.9 Phenylalanine Phe F Nonpolar Neutral 2.8 ProlinePro P Nonpolar Neutral −1.6 Serine Ser S Polar Neutral −0.8 ThreonineThr T Polar Neutral −0.7 Tryptophan Trp W Nonpolar Neutral −0.9 TyrosineTyr Y Polar Neutral −1.3 Valine Val V Nonpolar Neutral 4.2

A “homologous” sequence (e.g., nucleic acid sequence) includes asequence that is either identical or substantially similar to a knownreference sequence, such that it is, for example, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the knownreference sequence. Homologous sequences can include, for example,orthologous sequence and paralogous sequences. Homologous genes, forexample, typically descend from a common ancestral DNA sequence, eitherthrough a speciation event (orthologous genes) or a genetic duplicationevent (paralogous genes). “Orthologous” genes include genes in differentspecies that evolved from a common ancestral gene by speciation.Orthologs typically retain the same function in the course of evolution.“Paralogous” genes include genes related by duplication within a genome.Paralogs can evolve new functions in the course of evolution.

The term “in vitro” includes artificial environments and to processes orreactions that occur within an artificial environment (e.g., a test tubeor an isolated cell or cell line). The term “in vivo” includes naturalenvironments (e.g., a cell or organism or body) and to processes orreactions that occur within a natural environment. The term “ex vivo”includes cells that have been removed from the body of an individual andto processes or reactions that occur within such cells.

The term “reporter gene” refers to a nucleic acid having a sequenceencoding a gene product (typically an enzyme) that is easily andquantifiably assayed when a construct comprising the reporter genesequence operably linked to a heterologous promoter and/or enhancerelement is introduced into cells containing (or which can be made tocontain) the factors necessary for the activation of the promoter and/orenhancer elements. Examples of reporter genes include, but are notlimited, to genes encoding beta-galactosidase (lacZ), the bacterialchloramphenicol acetyltransferase (cat) genes, firefly luciferase genes,genes encoding beta-glucuronidase (GUS), and genes encoding fluorescentproteins. A “reporter protein” refers to a protein encoded by a reportergene.

The term “fluorescent reporter protein” as used herein means a reporterprotein that is detectable based on fluorescence wherein thefluorescence may be either from the reporter protein directly, activityof the reporter protein on a fluorogenic substrate, or a protein withaffinity for binding to a fluorescent tagged compound. Examples offluorescent proteins include green fluorescent proteins (e.g., GFP,GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g.,YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellowl), bluefluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv,Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP,Cerulean, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins(e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,mTangerine, and tdTomato), and any other suitable fluorescent proteinwhose presence in cells can be detected by flow cytometry methods.

Repair in response to double-strand breaks (DSBs) occurs principallythrough two conserved DNA repair pathways: homologous recombination (HR)and non-homologous end joining (NHEJ). See Kasparek & Humphrey (2011)Seminars in Cell & Dev. Biol. 22:886-897, herein incorporated byreference in its entirety for all purposes. Likewise, repair of a targetnucleic acid mediated by an exogenous donor nucleic acid can include anyprocess of exchange of genetic information between the twopolynucleotides.

The term “recombination” includes any process of exchange of geneticinformation between two polynucleotides and can occur by any mechanism.Recombination can occur via homology directed repair (HDR) or homologousrecombination (HR). HDR or HR includes a form of nucleic acid repairthat can require nucleotide sequence homology, uses a “donor” moleculeas a template for repair of a “target” molecule (i.e., the one thatexperienced the double-strand break), and leads to transfer of geneticinformation from the donor to target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or synthesis-dependent strand annealing, in which the donor is usedto resynthesize genetic information that will become part of the target,and/or related processes. In some cases, the donor polynucleotide, aportion of the donor polynucleotide, a copy of the donor polynucleotide,or a portion of a copy of the donor polynucleotide integrates into thetarget DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al.(2012) PLOS ONE 7:e45768:1-9; and Wang et al. (2013) Nat Biotechnol.31:530-532, each of which is herein incorporated by reference in itsentirety for all purposes.

NHEJ includes the repair of double-strand breaks in a nucleic acid bydirect ligation of the break ends to one another or to an exogenoussequence without the need for a homologous template. Ligation ofnon-contiguous sequences by NHEJ can often result in deletions,insertions, or translocations near the site of the double-strand break.For example, NHEJ can also result in the targeted integration of anexogenous donor nucleic acid through direct ligation of the break endswith the ends of the exogenous donor nucleic acid (i.e., NHEJ-basedcapture). Such NHEJ-mediated targeted integration can be preferred forinsertion of an exogenous donor nucleic acid when homology directedrepair (HDR) pathways are not readily usable (e.g., in non-dividingcells, primary cells, and cells which perform homology-based DNA repairpoorly). In addition, in contrast to homology-directed repair, knowledgeconcerning large regions of sequence identity flanking the cleavage siteis not needed, which can be beneficial when attempting targetedinsertion into organisms that have genomes for which there is limitedknowledge of the genomic sequence. The integration can proceed vialigation of blunt ends between the exogenous donor nucleic acid and thecleaved genomic sequence, or via ligation of sticky ends (i.e., having5′ or 3′ overhangs) using an exogenous donor nucleic acid that isflanked by overhangs that are compatible with those generated by anuclease agent in the cleaved genomic sequence. See, e.g., US2011/020722, WO 2014/033644, WO 2014/089290, and Maresca et al. (2013)Genome Res. 23(3):539-546, each of which is herein incorporated byreference in its entirety for all purposes. If blunt ends are ligated,target and/or donor resection may be needed to generation regions ofmicrohomology needed for fragment joining, which may create unwantedalterations in the target sequence.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” a protein maycontain the protein alone or in combination with other ingredients. Thetransitional phrase “consisting essentially of” means that the scope ofa claim is to be interpreted to encompass the specified elements recitedin the claim and those that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising.”

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur and that the description includesinstances in which the event or circumstance occurs and instances inwhich it does not.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

The term “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “or” refers to any one member of a particular list and alsoincludes any combination of members of that list.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a protein” or “at least one protein” can include a pluralityof proteins, including mixtures thereof.

Statistically significant means p≤0.05.

DETAILED DESCRIPTION

I. Overview

Cas-protein-ready tau biosensor cells and methods of making and usingsuch cells to screen for genetic modifiers of tau seeding or aggregationare provided. CRISPR/Cas synergistic activation mediator (SAM)-ready taubiosensor cells and methods of making and using such cells to screen forgenetic modifiers of tau seeding or aggregation are provided.Cas-protein-ready tau biosensor cells and methods of making and usingsuch cells to screen for genetic modifiers of tau disaggregation areprovided. CRISPR/Cas synergistic activation mediator (SAM)-ready taubiosensor cells and methods of making and using such cells to screen forgenetic modifiers of tau disaggregation are provided. Reagents andmethods for sensitizing such cells to tau seeding activity or tauaggregation are also provided. Reagents and methods for inducing tauaggregation are also provided.

To identify genes and pathways that modify the processes of abnormal tauprotein aggregation, a platform was developed for performing screenswith CRISPR (e.g., CRISPR/Cas9) nuclease (CRISPRn) sgRNA libraries toidentify genes that regulate the potential of cells to be “seeded” bytau disease-associated protein aggregates (e.g., genes which, whendisrupted, cause cells to be more susceptible to tau aggregate formationwhen exposed to a source of tau fibrillized protein). To furtheridentify genes and pathways that modify the processes of abnormal tauprotein aggregation, a platform was developed for performing screenswith CRISPR activation (CRISPRa) sgRNA libraries to identify genes thatregulate the potential of cells to be “seeded” by tau disease-associatedprotein aggregates (e.g., genes which, when transcriptionally activated,cause cells to be more susceptible to tau aggregate formation whenexposed to a source of tau fibrillized protein). Likewise, a platformwas developed for performing screens with CRISPR (e.g., CRISPR/Cas9)nuclease (CRISPRn) sgRNA libraries to identify genes that, whendisrupted, prevent tau aggregation or promote tau disaggregation.Likewise, a platform was developed for performing screens with CRISPRactivation (CRISPRa) sgRNA libraries to identify genes that, whentranscriptionally activated, prevent tau aggregation or promote taudisaggregation. A “seed” refers to one or more proteins that nucleateaggregation of other proteins with a similar aggregation domain. Theseeding activity of a sample refers to the ability of a sample tonucleate (i.e. induce) aggregation of a protein with a similaraggregation domain. The identification of such genes may elucidate themechanisms of tau cell-to-cell aggregate propagation and geneticpathways that govern the susceptibility of neurons to form tauaggregates in the context of neurodegenerative diseases.

The screens employ a tau biosensor cell line (e.g., human cell line, orHEK293T) consisting of cells stably expressing tau repeat domain (e.g.,tau four-repeat domain, tau4RD) with a pathogenic mutation (e.g., theP301S pathogenic mutation), linked to unique reporters that can acttogether as an intracellular biosensor that produces a detectable signalwhen aggregated. In one non-limiting example, the cell lines contain twotransgenes stably expressing disease-associated protein variants fusedto the fluorescent protein CFP (e.g., eCFP) or the fluorescent proteinYFP (e.g., eYFP): tau^(4RD)-CFP/tau^(4R)D-YFP (TCY), wherein the taurepeat domain (4RD) comprises the P301S pathogenic mutation. In thesebiosensor lines, tau-CFP/tau-YFP protein aggregation produces afluorescence resonance energy transfer (FRET) signal, the result of atransfer of fluorescent energy from donor CFP to acceptor YFP. The termCFP (cyan fluorescent protein) when used herein includes eCFP (enhancedcyan fluorescent protein), and the term YFP (yellow fluorescent protein)when used herein includes eYFP (enhanced yellow fluorescent protein).FRET-positive cells, which contain tau aggregates, can be sorted andisolated by flow cytometry. At baseline, unstimulated cells express thereporters in a stable, soluble state with minimal FRET signal. Uponstimulation (e.g., liposome transfection of seed particles), thereporter proteins form aggregates, producing a FRET signal.Aggregate-containing cells can be isolated by FACS. Stably propagatingaggregate-containing cell lines, Agg[+], can be isolated by clonalserial dilution of Agg[−] cell lines.

Several modifications were made to this tau biosensor cell line to makeit useful for genetic screening using CRISPRn libraries. First, thesetau biosensor cells were modified by introducing a Cas-expressingtransgene (e.g., Cas9 or SpCas9) for use in the CRISPRn screens. Second,reagents and a method were developed for sensitizing cells to tauseeding activity and tau aggregation. A cell line was developed in whichtau aggregates stably persist in all cells with growth and multiplepassages over time. These cells were used to produce conditioned mediumby collecting medium that has been on confluent cells for a period oftime. This conditioned medium can then be applied onto naïve taubiosensor tau cells at a ratio of so that tau aggregation could beinduced in a small percentage of these recipient cells, therebysensitizing them to tau seeding activity and tau aggregation.Conditioned medium without co-culturing has not been used in thiscontext as a seeding agent before. However, the conditioned medium isparticularly useful for large-scale genome-wide screens because taufibrils produced in vitro are a limited resource. In addition,conditioned medium is more physiologically relevant because it isproduced and secreted by cells rather than in vitro.

These cell lines were used to develop a method of screening in whichCas-expressing tau biosensor cells without aggregates (Agg[−]) weretransduced with a CRISPR guide RNA library to introduce knock-outmutations at each target gene. After culturing the cells to allow genomeediting and expansion, the cells were grown in conditioned medium tosensitize them to the seeding activity, and cells were identified inwhich tau aggregation occurred. Guide RNAs were identified that wereenriched in the aggregation-positive sub-population relative to earliertime points during genome editing and expansion to identify genes thatcan regulate the susceptibility of cells to tau seeding when exposed toan external source of tau seeding activity.

Likewise, several modifications were made to this tau biosensor cellline to make it useful for genetic screening using CRISPRa libraries(e.g., for use with a CRISPR/Cas synergistic activation mediator (SAM)system). In an exemplary SAM system, several activation domains interactto cause a greater transcriptional activation than could be induced byany one factor alone. For example, an exemplary SAM system comprises achimeric Cas protein comprising a nuclease-inactive Cas protein fused toone or more transcriptional activation domains (e.g., VP64) and achimeric adaptor protein comprising an adaptor protein (e.g., MS2 coatprotein (MCP)) fused to one or more transcriptional activation domains(e.g., fused to p65 and HSF1). The MCP naturally binds to MS2 stemloops. In an exemplary SAM system, MCP interacts MS2 stem loopsengineered into the CRISPR-associated sgRNA and thereby shuttles thebound transcription factors to the appropriate genomic location.

First, these tau biosensor cells were modified by introducing one ormore transgenes expressing the chimeric Cas protein comprising thenuclease-inactive Cas protein fused to one or more transcriptionalactivation domains (e.g., VP64) and the chimeric adaptor proteincomprising the adaptor protein (e.g., MS2 coat protein (MCP)) fused toone or more transcriptional activation domains (e.g., fused to p65 andHSF1). Although SAM systems are described herein, other CRISPRa systemssuch as a nuclease-inactive Cas protein fused to one or moretranscriptional activation domains, wherein such systems do not alsoinclude a chimeric adaptor protein, can also be used. In such cases, thetau biosensor cells would be modified by introducing a transgeneexpressing the chimeric Cas protein.

Second, reagents and a method were developed for sensitizing cells totau seeding activity and tau aggregation. A cell line was developed inwhich tau aggregates stably persist in all cells with growth andmultiple passages over time. These cells were used to produceconditioned medium by collecting medium that has been on confluent cellsfor a period of time. This conditioned medium can then be applied ontonaïve tau biosensor tau cells at a ratio of so that tau aggregationcould be induced in a small percentage of these recipient cells, therebysensitizing them to tau seeding activity and tau aggregation.Conditioned medium without co-culturing has not been used in thiscontext as a seeding agent before. However, the conditioned medium isparticularly useful for large-scale genome-wide screens because taufibrils produced in vitro are a limited resource. In addition,conditioned medium is more physiologically relevant because it isproduced and secreted by cells rather than in vitro.

These cell lines were used to develop a method of screening in whichSAM-expressing tau biosensor cells without aggregates (Agg[−]) weretransduced with a CRISPRa guide RNA library to transcriptionallyactivate each target gene. After culturing the cells to allow genomeediting and expansion, the cells were grown in conditioned medium tosensitize them to the seeding activity, and cells were identified inwhich tau aggregation occurred. Guide RNAs were identified that wereenriched in the aggregation-positive sub-population relative to earliertime points during genome editing and expansion to identify genes thatcan regulate the susceptibility of cells to tau seeding when exposed toan external source of tau seeding activity.

II. Cas/Tau Biosensor and SAM/Tau Biosensor Cell Lines and Methods ofGenerating

A. Cas/Tau Biosensor Cells and SAM/Tau Biosensor Cells

Disclosed herein are cells not only expressing a first tau repeat domain(e.g., comprising the tau microtubule binding domain (MBD)) linked to afirst reporter and a second tau repeat domain linked to a secondreporter, but also expressing a Cas protein, such as Cas9. Alsodisclosed herein are cells not only expressing a first tau repeat domain(e.g., comprising the tau microtubule binding domain (MBD)) linked to afirst reporter and a second tau repeat domain linked to a secondreporter, but also expressing a chimeric Cas protein comprising anuclease-inactive Cas protein fused to one or more transcriptionalactivation domains and a chimeric adaptor protein comprising an adaptorprotein fused to one or more transcriptional activation domains. Thefirst tau repeat domain linked to the first reporter can be stablyexpressed, and the second tau repeat domain linked to the secondreporter can be stably expressed. For example, DNA encoding the firsttau repeat domain linked to the first reporter can be genomicallyintegrated, and DNA encoding the second tau repeat domain linked to thesecond reporter can be genomically integrated. Similarly, the Casprotein can be stably expressed in the Cas/tau biosensor cells. Forexample, DNA encoding the Cas protein can be genomically integrated.Likewise, the chimeric Cas protein and/or the chimeric adaptor proteincan be stably expressed in the SAM/tau biosensor cells. For example, DNAencoding the chimeric Cas protein can be genomically integrated and/orDNA encoding the chimeric adaptor protein can be genomically integrated.The cells can be tau-aggregation-negative or can betau-aggregation-positive.

1. Tau and Tau Repeat Domains Linked to Reporters

Microtubule-associated protein tau is a protein that promotesmicrotubule assembly and stability and is predominantly expressed inneurons. Tau has a role in stabilizing neuronal microtubules and thus inpromoting axonal outgrowth. In Alzheimer's disease (AD) and a family ofrelated neurodegenerative diseases called tauopathies, tau protein isabnormally hyperphosphorylated and aggregated into bundles of filaments(paired helical filaments), which manifest as neurofibrillary tangles.Tauopathies are a group of heterogeneous neurodegenerative conditionscharacterized by deposition of abnormal tau in the brain.

The tau repeat domain can be from a tau protein from any animal ormammal, such as human, mouse, or rat. In one specific example, the taurepeat domain is from a human tau protein. An exemplary human tauprotein is assigned UniProt accession number P10636. The tau proteinsare the products of alternate splicing from a single gene that in humansis designated MAPT (microtubule-associated protein tau). The tau repeatdomain carries the sequence motifs responsible for aggregation (i.e., itis the aggregation-prone domain from tau). Depending on splicing, therepeat domain of the tau protein has either three or four repeat regionsthat constitute the aggregation-prone core of the protein, which isoften termed the repeat domain (RD). Specifically, the repeat domain oftau represents the core of the microtubule binding region and harborsthe hexapeptide motifs in R2 and R3 that are responsible for Tauaggregation. In the human brain, there are six tau isoforms ranging from352 to 441 amino acids in length. These isoforms vary at the carboxylterminal according to the presence of either three repeat or four repeatdomains (R1-R4), in addition to the presence or absence of one or twoinsert domains at the amino-terminus. The repeat domains, located at thecarboxyl-terminal half of tau, are believed to be important formicrotubule binding as well as for the pathological aggregation of tauinto paired helical filaments (PHFs), which are the core constituents ofthe neurofibrillary tangles found in tauopathies. Exemplary sequencesfor the four repeat domains (R1-R4) are provided in SEQ ID NOS: 1-4,respectively. Exemplary coding sequences for the four repeat domains(R1-R4) are provided in SEQ ID NOS: 5-8. An exemplary sequence for theTau four-repeat domain is provided in SEQ ID NO: 9. An exemplary codingsequence for the Tau four-repeat domain is provided in SEQ ID NO: 10. Anexemplary sequence for the Tau four-repeat domain with the P301Smutation is provided in SEQ ID NO: 11. An exemplary coding sequence forthe Tau four-repeat domain with the P301S mutation is provided in SEQ IDNO: 12.

The tau repeat domain used in the Cas/tau biosensor cells or the SAM/taubiosensor cells can comprise the tau microtubule binding domain (MBD).The tau repeat domain used in the Cas/tau biosensor cells or the SAM/taubiosensor cells can comprise one or more or all of the four repeatdomains (R1-R4). For example, the tau repeat domain can comprise,consist essentially of, or consist of one or more or all of SEQ ID NOS:1, 2, 3, and 4, or sequences at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS: 1, 2, 3, and 4. Inone specific example, the tau repeat domain is the tau four-repeatdomain (R1-R4) found in several tau isoforms. The tau four-repeat domaincan be used instead of full-length tau because it reliably forms fibrilsin cultured cells. For example, the tau repeat domain can comprise,consist essentially of, or consist of SEQ ID NO: 9 or SEQ ID NO: 11 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 9 or SEQ ID NO: 11. In one specific example,the nucleic acid encoding the tau repeat domain can comprise, consistessentially of, or consist of SEQ ID NO: 12 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 12, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 11.In another specific example, the nucleic acid encoding the second taurepeat domain linked to the second reporter can comprise, consistessentially of, or consist of SEQ ID NO: 10 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 10, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 9.The first and second tau repeat domains in the cells disclosed hereincan be the same, similar, or different.

One or both of the first tau repeat domain linked to the first reporterand the second tau repeat domain linked to the second reporter can bestably expressed in the cells. For example, nucleic acids encoding oneor both of the first tau repeat domain linked to the first reporter andthe second tau repeat domain linked to the second reporter can begenomically integrated in the population of cells and operably linked topromoters active in the cell.

The tau repeat domains used in the cells disclosed herein can alsocomprise a tau pathogenic mutation, such as a pro-aggregation mutation.Such a mutation can be, for example, a mutation that is associated with(e.g., segregates with) or causes a tauopathy. As one example, themutation can be an aggregation-sensitizing mutation that sensitizes tauto seeding but does not result in tau readily aggregating on its own.For example, the mutation can be the disease-associated P301S mutation.By P301S mutation is meant the human tau P301S mutation or acorresponding mutation in another tau protein when optimally alignedwith the human tau protein. The P301S mutation in tau exhibits highsensitivity to seeding, but it does not readily aggregate on its own.Thus, although at baseline tau reporter proteins comprising the P301Smutation exist in a stable, soluble form within the cell, exposure toexogenous tau seeds leads to tau reporter protein aggregation. Other taumutations include, for example, K280del, P301L, V337M, P301L/V337M, andK280del/I227P/I308P.

The first tau repeat domain can be linked to the first reporter and thesecond tau repeat domain can be linked to the second reporter by anymeans. For example, the reporter can be fused to the tau repeat domain(e.g., as part of a fusion protein).

The first reporter and second reporter can be and pair of uniquereporters that can act together as an intracellular biosensor thatproduces a detectable signal when the first and second proteins areaggregated. As one example, the reporters can be fluorescent proteins,and fluorescence resonance energy transfer (FRET) can be used to measureprotein aggregation. Specifically, the first and second reporters can bea FRET pair. Examples of FRET pairs (donor and acceptor fluorophores)are well known. See, e.g., Bajar et al. (2016) Sensors (Basel) 16(9):1488, herein incorporated by reference in its entirety for all purposes.Typical fluorescence microscopy techniques rely upon the absorption by afluorophore of light at one wavelength (excitation), followed by thesubsequent emission of secondary fluorescence at a longer wavelength.The mechanism of fluorescence resonance energy transfer involves a donorfluorophore in an excited electronic state, which may transfer itsexcitation energy to a nearby acceptor chromophore in a non-radiativefashion through long-range dipole-dipole interactions. For example, theFRET energy donor may be the first reporter, and the FRET energyacceptor may be the second reporter. Alternatively, the FRET energydonor may be the second reporter, and the FRET energy acceptor may bethe first reporter. In a specific example, the first and secondreporters are CFP and YFP. Exemplary protein and coding sequences forCFP are provided, e.g., in SEQ ID NOS: 13 and 14, respectively.Exemplary protein and coding sequences for YFP are provided, e.g., inSEQ ID NOS: 15 and 16, respectively. As a specific example, the CFP cancomprise, consist essentially of, or consist of SEQ ID NO: 13 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 13. As another specific example, the YFP cancomprise, consist essentially of, or consist of SEQ ID NO: 15 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 15.

As another example, a protein fragment complementation strategy can beused to detect aggregation. For example, a split-luciferase can be usedto produce bioluminescence from a substrate, and the first and secondreporters can be amino- (NLuc) and carboxy- (CLuc) terminal fragments ofthe luciferase. Examples of luciferase include Renilla, firefly, clickbeetle, and Metridia luciferase.

In one non-limiting example, the biosensor cells disclosed hereincontain two transgenes stably expressing disease-associated tau proteinvariants fused to the fluorescent protein CFP or the fluorescent proteinYFP, respectively (tau^(4RD)-CFP/tau^(4RD)-YFP (TCY)), wherein the taufour repeat domain (4RD) comprises the P301S pathogenic mutation. Inthese biosensor lines, tau-CFP/tau-YFP protein aggregation produces aFRET signal, the result of a transfer of fluorescent energy from donorCFP to acceptor YFP. FRET-positive cells, which contain tau aggregates,can be sorted and isolated by flow cytometry. At baseline, unstimulatedcells express the reporters in a stable, soluble state with minimal FRETsignal. Upon stimulation (e.g., liposome transfection of seedparticles), the reporter proteins form aggregates, producing a FRETsignal.

The Cas/tau biosensor cells disclosed herein can be aggregation-positive(Agg[+]) cells in which the tau repeat domain stably presents in anaggregated state, meaning that the tau repeat domain aggregates stablypersist in all cells with growth and multiple passages over time.Alternatively, the Cas/tau biosensor cells disclosed herein can beaggregation-negative (Agg[−]).

2. Cas Proteins and Chimeric Cas Proteins

The Cas/tau biosensor cells disclosed herein also comprise nucleic acids(DNA or RNA) encoding Cas proteins. Optionally, the Cas protein isstably expressed. Optionally, the cells comprise a genomicallyintegrated Cas coding sequence. Likewise, the SAM/tau biosensor cellsdisclosed herein also comprise nucleic acids (DNA or RNA) encodingchimeric Cas proteins comprising a nuclease-inactive Cas protein fusedto one or more transcriptional activation domains (e.g., VP64).Optionally, the chimeric Cas protein is stably expressed. Optionally,the cells comprise a genomically integrated chimeric Cas codingsequence.

Cas proteins are part of Clustered Regularly Interspersed ShortPalindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems. CRISPR/Cassystems include transcripts and other elements involved in theexpression of, or directing the activity of, Cas genes. A CRISPR/Cassystem can be, for example, a type I, a type II, a type III system, or atype V system (e.g., subtype V-A or subtype V-B). The methods andcompositions disclosed herein can employ CRISPR/Cas systems by utilizingCRISPR complexes (comprising a guide RNA (gRNA) complexed with a Casprotein) for site-directed binding or cleavage of nucleic acids.

CRISPR/Cas systems used in the compositions and methods disclosed hereincan be non-naturally occurring. A “non-naturally occurring” systemincludes anything indicating the involvement of the hand of man, such asone or more components of the system being altered or mutated from theirnaturally occurring state, being at least substantially free from atleast one other component with which they are naturally associated innature, or being associated with at least one other component with whichthey are not naturally associated. For example, some CRISPR/Cas systemsemploy non-naturally occurring CRISPR complexes comprising a gRNA and aCas protein that do not naturally occur together, employ a Cas proteinthat does not occur naturally, or employ a gRNA that does not occurnaturally.

Cas proteins generally comprise at least one RNA recognition or bindingdomain that can interact with guide RNAs. Cas proteins can also comprisenuclease domains (e.g., DNase domains or RNase domains), DNA-bindingdomains, helicase domains, protein-protein interaction domains,dimerization domains, and other domains. Some such domains (e.g., DNasedomains) can be from a native Cas protein. Other such domains can beadded to make a modified Cas protein. A nuclease domain possessescatalytic activity for nucleic acid cleavage, which includes thebreakage of the covalent bonds of a nucleic acid molecule. Cleavage canproduce blunt ends or staggered ends, and it can be single-stranded ordouble-stranded. For example, a wild type Cas9 protein will typicallycreate a blunt cleavage product. Alternatively, a wild type Cpf1 protein(e.g., FnCpf1) can result in a cleavage product with a 5-nucleotide 5′overhang, with the cleavage occurring after the 18th base pair from thePAM sequence on the non-targeted strand and after the 23rd base on thetargeted strand. A Cas protein can have full cleavage activity to createa double-strand break at a target genomic locus (e.g., a double-strandbreak with blunt ends), or it can be a nickase that creates asingle-strand break at a target genomic locus.

Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c,Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3,Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

An exemplary Cas protein is a Cas9 protein or a protein derived from aCas9 protein. Cas9 proteins are from a type II CRISPR/Cas system andtypically share four key motifs with a conserved architecture. Motifs 1,2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. ExemplaryCas9 proteins are from Streptococcus pyogenes, Streptococcusthermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsisdassonvillei, Streptomyces pristinaespiralis, Streptomycesviridochromogenes, Streptomyces viridochromogenes, Streptosporangiumroseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius,Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacteriumsibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius,Microscilla marina, Burkholderiales bacterium, Polaromonasnaphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotogamobilis, Thermosipho africanus, Acaryochloris marina, Neisseriameningitidis, or Campylobacter jejuni. Additional examples of the Cas9family members are described in WO 2014/131833, herein incorporated byreference in its entirety for all purposes. Cas9 from S. pyogenes(SpCas9) (assigned SwissProt accession number Q99ZW2) is an exemplaryCas9 protein. Exemplary SpCas9 protein and coding sequence are set forthin SEQ ID NOS: 21 and 22, respectively. Cas9 from S. aureus (SaCas9)(assigned UniProt accession number J7RUA5) is another exemplary Cas9protein. Cas9 from Campylobacter jejuni (CjCas9) (assigned UniProtaccession number Q0P897) is another exemplary Cas9 protein. See, e.g.,Kim et al. (2017) Nat. Comm. 8:14500, herein incorporated by referencein its entirety for all purposes. SaCas9 is smaller than SpCas9, andCjCas9 is smaller than both SaCas9 and SpCas9. Cas9 from Neisseriameningitidis (Nme2Cas9) is another exemplary Cas9 protein. See, e.g.,Edraki et al. (2019) Mol. Cell 73(4):714-726, herein incorporated byreference in its entirety for all purposes. Cas9 proteins fromStreptococcus thermophilus (e.g., Streptococcus thermophilus LMD-9 Cas9encoded by the CRISPR1 locus (St1Cas9) or Streptococcus thermophilusCas9 from the CRISPR3 locus (St3Cas9)) are other exemplary Cas9proteins. Cas9 from Francisella novicida (FnCas9) or the RHA Francisellanovicida Cas9 variant that recognizes an alternative PAM(E1369R/E1449H/R1556A substitutions) are other exemplary Cas9 proteins.These and other exemplary Cas9 proteins are reviewed, e.g., inCebrian-Serrano and Davies (2017) Mamm. Genome 28(7):247-261, hereinincorporated by reference in its entirety for all purposes.

As one example, the Cas protein can be a Cas9 protein. For example, theCas9 protein can be a Streptococcus pyogenes Cas9 protein. As onespecific example, the Cas protein can comprise, consist essentially of,or consist of SEQ ID NO: 21 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21. Asanother specific example, a chimeric Cas protein comprising anuclease-inactive Cas protein and one or more transcriptional activationdomains can comprise, consist essentially of, or consist of SEQ ID NO:36 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NO: 36.

Another example of a Cas protein is a Cpf1 (CRISPR from Prevotella andFrancisella 1) protein. Cpf1 is a large protein (about 1300 amino acids)that contains a RuvC-like nuclease domain homologous to thecorresponding domain of Cas9 along with a counterpart to thecharacteristic arginine-rich cluster of Cas9. However, Cpf1 lacks theHNH nuclease domain that is present in Cas9 proteins, and the RuvC-likedomain is contiguous in the Cpf1 sequence, in contrast to Cas9 where itcontains long inserts including the HNH domain. See, e.g., Zetsche etal. (2015) Cell 163(3):759-771, herein incorporated by reference in itsentirety for all purposes. Exemplary Cpf1 proteins are from Francisellatularensis 1, Francisella tularensis subsp. novicida, Prevotellaalbensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011 GWA2_33_10,Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC,Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, CandidatusMethanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237,Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonascrevioricanis 3, Prevotella disiens, and Porphyromonas macacae. Cpf1from Francisella novicida U112 (FnCpf1; assigned UniProt accessionnumber A0Q7Q2) is an exemplary Cpf1 protein.

Cas proteins can be wild type proteins (i.e., those that occur innature), modified Cas proteins (i.e., Cas protein variants), orfragments of wild type or modified Cas proteins. Cas proteins can alsobe active variants or fragments with respect to catalytic activity ofwild type or modified Cas proteins. Active variants or fragments withrespect to catalytic activity can comprise at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thewild type or modified Cas protein or a portion thereof, wherein theactive variants retain the ability to cut at a desired cleavage site andhence retain double-strand-break-inducing activity. Assays fordouble-strand-break-inducing activity are known and generally measurethe overall activity and specificity of the Cas protein on DNAsubstrates containing the cleavage site.

One example of a modified Cas protein is the modified SpCas9-HF1protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9harboring alterations (N497A/R661A/Q695A/Q926A) designed to reducenon-specific DNA contacts. See, e.g., Kleinstiver et al. (2016) Nature529(7587):490-495, herein incorporated by reference in its entirety forall purposes. Another example of a modified Cas protein is the modifiedeSpCas9 variant (K848A/K1003A/R1060A) designed to reduce off-targeteffects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88,herein incorporated by reference in its entirety for all purposes. OtherSpCas9 variants include K855A and K810A/K1003A/R1060A. These and othermodified Cas proteins are reviewed, e.g., in Cebrian-Serrano and Davies(2017) Mamm. Genome 28(7):247-261, herein incorporated by reference inits entirety for all purposes. Another example of a modified Cas9protein is xCas9, which is a SpCas9 variant that can recognize anexpanded range of PAM sequences. See, e.g., Hu et al. (2018) Nature556:57-63, herein incorporated by reference in its entirety for allpurposes.

Cas proteins can be modified to increase or decrease one or more ofnucleic acid binding affinity, nucleic acid binding specificity, andenzymatic activity. Cas proteins can also be modified to change anyother activity or property of the protein, such as stability. Forexample, a Cas protein can be truncated to remove domains that are notessential for the function of the protein or to optimize (e.g., enhanceor reduce) the activity of or a property of the Cas protein. As anotherexample, one or more nuclease domains of the Cas protein can bemodified, deleted, or inactivated (e.g., for use in the SAM/taubiosensor cells comprising a nuclease-inactive Cas protein).

Cas proteins can comprise at least one nuclease domain, such as a DNasedomain. For example, a wild type Cpf1 protein generally comprises aRuvC-like domain that cleaves both strands of target DNA, perhaps in adimeric configuration. Cas proteins can also comprise at least twonuclease domains, such as DNase domains. For example, a wild type Cas9protein generally comprises a RuvC-like nuclease domain and an HNH-likenuclease domain. The RuvC and HNH domains can each cut a differentstrand of double-stranded DNA to make a double-stranded break in theDNA. See, e.g., Jinek et al. (2012) Science 337:816-821, hereinincorporated by reference in its entirety for all purposes.

One or more or all of the nuclease domains can be deleted or mutated sothat they are no longer functional or have reduced nuclease activity.For example, if one of the nuclease domains is deleted or mutated in aCas9 protein, the resulting Cas9 protein can be referred to as a nickaseand can generate a single-strand break within a double-stranded targetDNA but not a double-strand break (i.e., it can cleave the complementarystrand or the non-complementary strand, but not both). If both of thenuclease domains are deleted or mutated, the resulting Cas protein(e.g., Cas9) will have a reduced ability to cleave both strands of adouble-stranded DNA (e.g., a nuclease-null or nuclease-inactive Casprotein, or a catalytically dead Cas protein (dCas)). An example of amutation that converts Cas9 into a nickase is a D10A (aspartate toalanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 fromS. pyogenes. Likewise, H939A (histidine to alanine at amino acidposition 839), H840A (histidine to alanine at amino acid position 840),or N863A (asparagine to alanine at amino acid position N863) in the HNHdomain of Cas9 from S. pyogenes can convert the Cas9 into a nickase.Other examples of mutations that convert Cas9 into a nickase include thecorresponding mutations to Cas9 from S. thermophilus. See, e.g.,Sapranauskas et al. (2011) Nucleic Acids Res. 39(21):9275-9282 and WO2013/141680, each of which is herein incorporated by reference in itsentirety for all purposes. Such mutations can be generated using methodssuch as site-directed mutagenesis, PCR-mediated mutagenesis, or totalgene synthesis. Examples of other mutations creating nickases can befound, for example, in WO 2013/176772 and WO 2013/142578, each of whichis herein incorporated by reference in its entirety for all purposes. Ifall of the nuclease domains are deleted or mutated in a Cas protein(e.g., both of the nuclease domains are deleted or mutated in a Cas9protein), the resulting Cas protein (e.g., Cas9) will have a reducedability to cleave both strands of a double-stranded DNA (e.g., anuclease-null or nuclease-inactive Cas protein). One specific example isa D10A/H840A S. pyogenes Cas9 double mutant or a corresponding doublemutant in a Cas9 from another species when optimally aligned with S.pyogenes Cas9. Another specific example is a D10A/N863A S. pyogenes Cas9double mutant or a corresponding double mutant in a Cas9 from anotherspecies when optimally aligned with S. pyogenes Cas9.

Examples of inactivating mutations in the catalytic domains of xCas9 arethe same as those described above for SpCas9. Examples of inactivatingmutations in the catalytic domains of Staphylococcus aureus Cas9proteins are also known. For example, the Staphylococcus aureus Cas9enzyme (SaCas9) may comprise a substitution at position N580 (e.g.,N580A substitution) and a substitution at position D10 (e.g., D10Asubstitution) to generate a nuclease-inactive Cas protein. See, e.g., WO2016/106236, herein incorporated by reference in its entirety for allpurposes. Examples of inactivating mutations in the catalytic domains ofNme2Cas9 are also known (e.g., combination of D16A and H588A). Examplesof inactivating mutations in the catalytic domains of St1Cas9 are alsoknown (e.g., combination of D9A, D598A, H599A, and N622A). Examples ofinactivating mutations in the catalytic domains of St3Cas9 are alsoknown (e.g., combination of D10A and N870A). Examples of inactivatingmutations in the catalytic domains of CjCas9 are also known (e.g.,combination of D8A and H559A). Examples of inactivating mutations in thecatalytic domains of FnCas9 and RHA FnCas9 are also known (e.g., N995A).

Examples of inactivating mutations in the catalytic domains of Cpf1proteins are also known. With reference to Cpf1 proteins fromFrancisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1),Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella bovoculi 237(MbCpf1 Cpf1), such mutations can include mutations at positions 908,993, or 1263 of AsCpf1 or corresponding positions in Cpf1 orthologs, orpositions 832, 925, 947, or 1180 of LbCpf1 or corresponding positions inCpf1 orthologs. Such mutations can include, for example one or more ofmutations D908A, E993A, and D1263A of AsCpf1 or corresponding mutationsin Cpf1 orthologs, or D832A, E925A, D947A, and D1180A of LbCpf1 orcorresponding mutations in Cpf1 orthologs. See, e.g., US 2016/0208243,herein incorporated by reference in its entirety for all purposes.

Cas proteins can also be operably linked to heterologous polypeptides asfusion proteins. For example, a Cas protein can be fused to a cleavagedomain, an epigenetic modification domain, a transcriptional activationdomain, or a transcriptional repressor domain. See WO 2014/089290,herein incorporated by reference in its entirety for all purposes. Forexample, Cas proteins can be operably linked or fused to atranscriptional activation domain for use in the SAM/tau biosensorcells. Examples of transcriptional activation domains include a herpessimplex virus VP16 activation domain, VP64 (which is a tetramericderivative of VP16), a NFκB p65 activation domain, p53 activationdomains 1 and 2, a CREB (cAMP response element binding protein)activation domain, an E2A activation domain, and an NFAT (nuclear factorof activated T-cells) activation domain. Other examples includeactivation domains from Oct1, Oct-2A, SP1, AP-2, CTF1, P300, CBP, PCAF,SRC1, PvALF, ERF-2, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7, ARF-8,CPRF1, CPRF4, MYC-RP/GP, TRAB1PC4, and HSF1. See, e.g., US 2016/0237456,EP3045537, and WO 2011/146121, each of which is incorporated byreference in its entirety for all purposes. In some cases, atranscriptional activation system can be used comprising a dCas9-VP64fusion protein paired with MS2-p65-HSF1. Guide RNAs in such systems canbe designed with aptamer sequences appended to sgRNA tetraloop andstem-loop 2 designed to bind dimerized MS2 bacteriophage coat proteins.See, e.g., Konermann et al. (2015) Nature 517(7536):583-588, hereinincorporated by reference in its entirety for all purposes. Examples oftranscriptional repressor domains include inducible cAMP early repressor(ICER) domains, Kruppel-associated box A (KRAB-A) repressor domains, YY1glycine rich repressor domains, Sp1-like repressors, E(spl) repressors,IκB repressor, and MeCP2. Other examples include transcriptionalrepressor domains from A/B, KOX, TGF-beta-inducible early gene (TIEG),v-erbA, SID, SID4X, MBD2, MBD3, DNMT1, DNMG3A, DNMT3B, Rb, ROM2, See,e.g., EP3045537 and WO 2011/146121, each of which is incorporated byreference in its entirety for all purposes. Cas proteins can also befused to a heterologous polypeptide providing increased or decreasedstability. The fused domain or heterologous polypeptide can be locatedat the N-terminus, the C-terminus, or internally within the Cas protein.

Cas proteins can also be operably linked to heterologous polypeptides asfusion proteins. As one example, a Cas protein can be fused to one ormore heterologous polypeptides that provide for subcellularlocalization. Such heterologous polypeptides can include, for example,one or more nuclear localization signals (NLS) such as the monopartiteSV40 NLS and/or a bipartite alpha-importin NLS for targeting to thenucleus, a mitochondrial localization signal for targeting to themitochondria, an ER retention signal, and the like. See, e.g., Lange etal. (2007)J. Biol. Chem. 282:5101-5105, herein incorporated by referencein its entirety for all purposes. Such subcellular localization signalscan be located at the N-terminus, the C-terminus, or anywhere within theCas protein. An NLS can comprise a stretch of basic amino acids, and canbe a monopartite sequence or a bipartite sequence. Optionally, a Casprotein can comprise two or more NLSs, including an NLS (e.g., analpha-importin NLS or a monopartite NLS) at the N-terminus and an NLS(e.g., an SV40 NLS or a bipartite NLS) at the C-terminus. A Cas proteincan also comprise two or more NLSs at the N-terminus and/or two or moreNLSs at the C-terminus.

Cas proteins can also be operably linked to a cell-penetrating domain orprotein transduction domain. For example, the cell-penetrating domaincan be derived from the HIV-1 TAT protein, the TLM cell-penetratingmotif from human hepatitis B virus, MPG, Pep-1, VP22, a cell penetratingpeptide from Herpes simplex virus, or a polyarginine peptide sequence.See, e.g., WO 2014/089290 and WO 2013/176772, each of which is hereinincorporated by reference in its entirety for all purposes. Thecell-penetrating domain can be located at the N-terminus, theC-terminus, or anywhere within the Cas protein.

Cas proteins can also be operably linked to a heterologous polypeptidefor ease of tracking or purification, such as a fluorescent protein, apurification tag, or an epitope tag. Examples of fluorescent proteinsinclude green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP,eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP,ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus,YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2,Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescentproteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), redfluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer,mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem,HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orangefluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato), and any other suitablefluorescent protein. Examples of tags include glutathione-S-transferase(GST), chitin binding protein (CBP), maltose binding protein,thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag,myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G,histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.

Cas proteins can be provided in any form. For example, a Cas protein canbe provided in the form of a protein. For example, a Cas protein can beprovided as a Cas protein complexed with a gRNA. Alternatively, a Casprotein can be provided in the form of a nucleic acid encoding the Casprotein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA. Optionally,the nucleic acid encoding the Cas protein can be codon optimized forefficient translation into protein in a particular cell or organism. Forexample, the nucleic acid encoding the Cas protein can be modified tosubstitute codons having a higher frequency of usage in a bacterialcell, a yeast cell, a human cell, a non-human cell, a mammalian cell, arodent cell, a mouse cell, a rat cell, or any other host cell ofinterest, as compared to the naturally occurring polynucleotidesequence. For example, the nucleic acid encoding the Cas protein can becodon optimized for expression in a human cell. When a nucleic acidencoding the Cas protein is introduced into the cell, the Cas proteincan be transiently, conditionally, or constitutively expressed in thecell.

Cas proteins provided as mRNAs can be modified for improved stabilityand/or immunogenicity properties. The modifications may be made to oneor more nucleosides within the mRNA. Examples of chemical modificationsto mRNA nucleobases include pseudouridine, 1-methyl-pseudouridine, and5-methyl-cytidine. For example, capped and polyadenylated Cas mRNAcontaining N1-methyl pseudouridine can be used. Likewise, Cas mRNAs canbe modified by depletion of uridine using synonymous codons.

Nucleic acids encoding Cas proteins can be stably integrated in thegenome of a cell and operably linked to a promoter active in the cell.In one specific example, the nucleic acid encoding the Cas protein cancomprise, consist essentially of, or consist of SEQ ID NO: 22 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 22, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 21. In another specific example, the nucleic acid encodinga chimeric Cas protein comprising a nuclease-inactive Cas protein andone or more transcriptional activation domains can comprise, consistessentially of, or consist of SEQ ID NO: 38 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 38, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 36.Alternatively, nucleic acids encoding Cas proteins can be operablylinked to a promoter in an expression construct. Expression constructsinclude any nucleic acid constructs capable of directing expression of agene or other nucleic acid sequence of interest (e.g., a Cas gene) andwhich can transfer such a nucleic acid sequence of interest to a targetcell. Promoters that can be used in an expression construct includepromoters active, for example, in one or more of a eukaryotic cell, ahuman cell, a non-human cell, a mammalian cell, a non-human mammaliancell, a rodent cell, a mouse cell, a rat cell, a pluripotent cell, anembryonic stem (ES) cell, an adult stem cell, a developmentallyrestricted progenitor cell, an induced pluripotent stem (iPS) cell, or aone-cell stage embryo. Such promoters can be, for example, conditionalpromoters, inducible promoters, constitutive promoters, ortissue-specific promoters.

3. Chimeric Adaptor Proteins

The SAM/tau biosensor cells disclosed herein can comprise not onlynucleic acids (DNA or RNA) encoding a chimeric Cas protein comprising anuclease-inactive Cas protein fused to one or more transcriptionalactivation domains (e.g., VP64) but optionally also nucleic acids (DNAor RNA) encoding a chimeric adaptor protein comprising an adaptorprotein (e.g., MS2 coat protein (MCP)) fused to one or moretranscriptional activation domains (e.g., fused to p65 and HSF1).Optionally, the chimeric Cas protein and/or the chimeric adaptor proteinis stably expressed. Optionally, the cells comprise a genomicallyintegrated chimeric Cas protein coding sequence and/or a genomicallyintegrated chimeric adaptor protein coding sequence.

Such chimeric adaptor proteins comprise: (a) an adaptor (i.e., adaptordomain or adaptor protein) that specifically binds to an adaptor-bindingelement within a guide RNA; and (b) one or more heterologoustranscriptional activation domains. For example, such fusion proteinscan comprise 1, 2, 3, 4, 5, or more transcriptional activation domains(e.g., two or more heterologous transcriptional activation domains orthree or more heterologous transcriptional activation domains). In oneexample, such chimeric adaptor proteins can comprise: (a) an adaptor(i.e., an adaptor domain or adaptor protein) that specifically binds toan adaptor-binding element in a guide RNA; and (b) two or moretranscriptional activation domains. For example, the chimeric adaptorprotein can comprise: (a) an MS2 coat protein adaptor that specificallybinds to one or more MS2 aptamers in a guide RNA (e.g., two MS2 aptamersin separate locations in a guide RNA); and (b) one or more (e.g., two ormore transcriptional activation domains). For example, the twotranscriptional activation domains can be p65 and HSF1 transcriptionalactivation domains or functional fragments or variants thereof. However,chimeric adaptor proteins in which the transcriptional activationdomains comprise other transcriptional activation domains or functionalfragments or variants thereof are also provided.

The one or more transcriptional activation domains can be fused directlyto the adaptor. Alternatively, the one or more transcriptionalactivation domains can be linked to the adaptor via a linker or acombination of linkers or via one or more additional domains. Likewise,if two or more transcriptional activation domains are present, they canbe fused directly to each other or can be linked to each other via alinker or a combination of linkers or via one or more additionaldomains. Linkers that can be used in these fusion proteins can includeany sequence that does not interfere with the function of the fusionproteins. Exemplary linkers are short (e.g., 2-20 amino acids) and aretypically flexible (e.g., comprising amino acids with a high degree offreedom such as glycine, alanine, and serine).

The one or more transcriptional activation domains and the adaptor canbe in any order within the chimeric adaptor protein. As one option, theone or more transcriptional activation domains can be C-terminal to theadaptor and the adaptor can be N-terminal to the one or moretranscriptional activation domains. For example, the one or moretranscriptional activation domains can be at the C-terminus of thechimeric adaptor protein, and the adaptor can be at the N-terminus ofthe chimeric adaptor protein. However, the one or more transcriptionalactivation domains can be C-terminal to the adaptor without being at theC-terminus of the chimeric adaptor protein (e.g., if a nuclearlocalization signal is at the C-terminus of the chimeric adaptorprotein). Likewise, the adaptor can be N-terminal to the one or moretranscriptional activation domains without being at the N-terminus ofthe chimeric adaptor protein (e.g., if a nuclear localization signal isat the N-terminus of the chimeric adaptor protein). As another option,the one or more transcriptional activation domains can be N-terminal tothe adaptor and the adaptor can be C-terminal to the one or moretranscriptional activation domains. For example, the one or moretranscriptional activation domains can be at the N-terminus of thechimeric adaptor protein, and the adaptor can be at the C-terminus ofthe chimeric adaptor protein. As yet another option, if the chimericadaptor protein comprises two or more transcriptional activationdomains, the two or more transcriptional activation domains can flankthe adaptor.

Chimeric adaptor proteins can also be operably linked or fused toadditional heterologous polypeptides. The fused or linked heterologouspolypeptide can be located at the N-terminus, the C-terminus, oranywhere internally within the chimeric adaptor protein. For example, achimeric adaptor protein can further comprise a nuclear localizationsignal. A specific example of such a protein comprises an MS2 coatprotein (adaptor) linked (either directly or via an NLS) to a p65transcriptional activation domain C-terminal to the MS2 coat protein(MCP), and HSF1 transcriptional activation domain C-terminal to the p65transcriptional activation domain. Such a protein can comprise fromN-terminus to C-terminus: an MCP; a nuclear localization signal; a p65transcriptional activation domain; and an HSF1 transcriptionalactivation domain. For example, a chimeric adaptor protein can comprise,consist essentially of, or consist of an amino acid sequence at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the MCP-p65-HSF1 chimeric adaptor protein sequence set forth in SEQID NO: 37. Likewise, a nucleic acid encoding a chimeric adaptor proteincan comprise, consist essentially of, or consist of a sequence at least85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the MCP-p65-HSF1 chimeric adaptor protein coding sequence set forthin SEQ ID NO: 39

Adaptors (i.e., adaptor domains or adaptor proteins) arenucleic-acid-binding domains (e.g., DNA-binding domains and/orRNA-binding domains) that specifically recognize and bind to distinctsequences (e.g., bind to distinct DNA and/or RNA sequences such asaptamers in a sequence-specific manner). Aptamers include nucleic acidsthat, through their ability to adopt a specific three-dimensionalconformation, can bind to a target molecule with high affinity andspecificity. Such adaptors can bind, for example, to a specific RNAsequence and secondary structure. These sequences (i.e., adaptor-bindingelements) can be engineered into a guide RNA. For example, an MS2aptamer can be engineered into a guide RNA to specifically bind an MS2coat protein (MCP). For example, the adaptor can comprise, consistessentially of, or consist of an amino acid sequence at least 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theMCP sequence set forth in SEQ ID NO: 40. Likewise, a nucleic acidencoding the adaptor can comprise, consist essentially of, or consist ofan amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the MCP coding sequence set forth inSEQ ID NO: 41. Specific examples of adaptors and targets include, forexample, RNA-binding protein/aptamer combinations that exist within thediversity of bacteriophage coat proteins. See, e.g., US 2019-0284572 andWO 2019/183123, each of which is herein incorporated by reference in itsentirety for all purposes.

The chimeric adaptor proteins disclosed herein comprise one or moretranscriptional activation domains. Such transcriptional activationdomains can be naturally occurring transcriptional activation domains,can be functional fragments or functional variants of naturallyoccurring transcriptional activation domains, or can be engineered orsynthetic transcriptional activation domains. Transcriptional activationdomains that can be used include those described, for example, in US2019-0284572 and WO 2019/183123, each of which is herein incorporated byreference in its entirety for all purposes.

4. Cell Types

The Cas/tau biosensor cells disclosed herein can be any type of cell andcan be in vitro, ex vivo, or in vivo. A Cas/tau biosensor cell line orpopulation of cells can be a monoclonal cell line or population ofcells. Likewise, the SAM/tau biosensor cells disclosed herein can be anytype of cell and can be in vitro, ex vivo, or in vivo. A SAM/taubiosensor cell line or population of cells can be a monoclonal cell lineor population of cells. The cell can be from any source. For example,the cell can be a eukaryotic cell, an animal cell, a plant cell, or afungal (e.g., yeast) cell. Such cells can be fish cells or bird cells,or such cells can be mammalian cells, such as human cells, non-humanmammalian cells, rodent cells, mouse cells, or rat cells. Mammalsinclude, for example, humans, non-human primates, monkeys, apes, catsdogs, horses, bulls, deer, bison, sheep, rodents (e.g., mice, rats,hamsters, guinea pigs), livestock (e.g., bovine species such as cows andsteer; ovine species such as sheep and goats; and porcine species suchas pigs and boars). Birds include, for example, chickens, turkeys,ostrich, geese, and ducks. Domesticated animals and agricultural animalsare also included. The term “non-human animal” excludes humans. In aspecific example, the Cas/tau biosensor cells are human cells (e.g.,HEK293T cells). Likewise, in a specific example, the SAM/tau biosensorcells are human cells (e.g., HEK293T cells).

The cell can be, for example, a totipotent cell or a pluripotent cell(e.g., an embryonic stem (ES) cell such as a rodent ES cell, a mouse EScell, or a rat ES cell). Totipotent cells include undifferentiated cellsthat can give rise to any cell type, and pluripotent cells includeundifferentiated cells that possess the ability to develop into morethan one differentiated cell types. Such pluripotent and/or totipotentcells can be, for example, ES cells or ES-like cells, such as an inducedpluripotent stem (iPS) cells. ES cells include embryo-derived totipotentor pluripotent cells that are capable of contributing to any tissue ofthe developing embryo upon introduction into an embryo. ES cells can bederived from the inner cell mass of a blastocyst and are capable ofdifferentiating into cells of any of the three vertebrate germ layers(endoderm, ectoderm, and mesoderm).

The cell can also be a primary somatic cell, or a cell that is not aprimary somatic cell. Somatic cells can include any cell that is not agamete, germ cell, gametocyte, or undifferentiated stem cell. The cellcan also be a primary cell. Primary cells include cells or cultures ofcells that have been isolated directly from an organism, organ, ortissue. Primary cells include cells that are neither transformed norimmortal. They include any cell obtained from an organism, organ, ortissue which was not previously passed in tissue culture or has beenpreviously passed in tissue culture but is incapable of beingindefinitely passed in tissue culture. Such cells can be isolated byconventional techniques and include, for example, somatic cells,hematopoietic cells, endothelial cells, epithelial cells, fibroblasts,mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclearcells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes,skeletal myoblasts, and smooth muscle cells. For example, primary cellscan be derived from connective tissues, muscle tissues, nervous systemtissues, or epithelial tissues.

Such cells also include would normally not proliferate indefinitely but,due to mutation or alteration, have evaded normal cellular senescenceand instead can keep undergoing division. Such mutations or alterationscan occur naturally or be intentionally induced. Examples ofimmortalized cells include Chinese hamster ovary (CHO) cells, humanembryonic kidney cells (e.g., HEK293T cells), and mouse embryonicfibroblast cells (e.g., 3T3 cells). Numerous types of immortalized cellsare well known. Immortalized or primary cells include cells that aretypically used for culturing or for expressing recombinant genes orproteins.

The cell can also be a differentiated cell, such as a neuronal cell(e.g., a human neuronal cell).

B. Methods of Generating Cas/Tau Biosensor Cells and SAM/Tau BiosensorCells

The Cas/tau biosensor cells disclosed herein can be generated by anyknown means. The first tau repeat domain linked to the first reporter,the second tau repeat domain linked to the second reporter, and the Casprotein can be introduced into the cell in any form (e.g., DNA, RNA, orprotein) by any known means. Likewise, the SAM/tau biosensor cellsdisclosed herein can be generated by any known means. The first taurepeat domain linked to the first reporter, the second tau repeat domainlinked to the second reporter, the chimeric Cas protein, and thechimeric adaptor protein can be introduced into the cell in any form(e.g., DNA, RNA, or protein) by any known means. “Introducing” includespresenting to the cell the nucleic acid or protein in such a manner thatthe sequence gains access to the interior of the cell. The methodsprovided herein do not depend on a particular method for introducing anucleic acid or protein into the cell, only that the nucleic acid orprotein gains access to the interior of a least one cell. Methods forintroducing nucleic acids and proteins into various cell types are knownand include, for example, stable transfection methods, transienttransfection methods, and virus-mediated methods. Optionally, targetingvectors can be used.

Transfection protocols as well as protocols for introducing nucleicacids or proteins into cells may vary. Non-limiting transfection methodsinclude chemical-based transfection methods using liposomes;nanoparticles; calcium phosphate (Graham et al. (1973) Virology 52 (2):456-67, Bacchetti et al. (1977) Proc. Natl. Acad. Sci. USA 74 (4):1590-4, and Kriegler, M (1991). Transfer and Expression: A LaboratoryManual. New York: W. H. Freeman and Company. pp. 96-97); dendrimers; orcationic polymers such as DEAE-dextran or polyethylenimine. Non-chemicalmethods include electroporation, Sono-poration, and opticaltransfection. Particle-based transfection includes the use of a genegun, or magnet-assisted transfection (Bertram (2006) CurrentPharmaceutical Biotechnology 7, 277-28). Viral methods can also be usedfor transfection.

Introduction of nucleic acids or proteins into a cell can also bemediated by electroporation, by intracytoplasmic injection, by viralinfection, by adenovirus, by adeno-associated virus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection, or bynucleofection. Nucleofection is an improved electroporation technologythat enables nucleic acid substrates to be delivered not only to thecytoplasm but also through the nuclear membrane and into the nucleus. Inaddition, use of nucleofection in the methods disclosed herein typicallyrequires much fewer cells than regular electroporation (e.g., only about2 million compared with 7 million by regular electroporation). In oneexample, nucleofection is performed using the LONZA® NUCLEOFECTOR™system.

Introduction of nucleic acids or proteins into a cell can also beaccomplished by microinjection. Microinjection of an mRNA is preferablyinto the cytoplasm (e.g., to deliver mRNA directly to the translationmachinery), while microinjection of a protein or a DNA encoding aprotein is preferably into the nucleus. Alternatively, microinjectioncan be carried out by injection into both the nucleus and the cytoplasm:a needle can first be introduced into the nucleus and a first amount canbe injected, and while removing the needle from the cell a second amountcan be injected into the cytoplasm. Methods for carrying outmicroinjection are well known. See, e.g., Nagy et al. (Nagy A,Gertsenstein M, Vintersten K, Behringer R., 2003, Manipulating the MouseEmbryo. Cold Spring Harbor, New York: Cold Spring Harbor LaboratoryPress); Meyer et al. (2010) Proc. Natl. Acad. Sci. USA 107:15022-15026and Meyer et al. (2012) Proc. Natl. Acad. Sci. USA 109:9354-9359.

Other methods for introducing nucleic acid or proteins into a cell caninclude, for example, vector delivery, particle-mediated delivery,exosome-mediated delivery, lipid-nanoparticle-mediated delivery,cell-penetrating-peptide-mediated delivery, orimplantable-device-mediated delivery. Methods of administering nucleicacids or proteins to a subject to modify cells in vivo are disclosedelsewhere herein.

In one example, the first tau repeat domain linked to the firstreporter, the second tau repeat domain linked to the second reporter,and the Cas protein can be introduced via viral transduction such aslentiviral transduction.

Screening for cells comprising the first tau repeat domain linked to thefirst reporter, the second tau repeat domain linked to the secondreporter, and the Cas protein can be performed by any known means.

As one example, reporter genes can be used to screen for cells that havethe Cas protein, the first tau repeat domain linked to the firstreporter, or the second tau repeat domain linked to the second reporter.Exemplary reporter genes include those encoding luciferase,3-galactosidase, green fluorescent protein (GFP), enhanced greenfluorescent protein (eGFP), cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP),blue fluorescent protein (BFP), enhanced blue fluorescent protein(eBFP), DsRed, ZsGreen, MmGFP, mPlum, mCherry, tdTomato, mStrawberry,J-Red, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, Cerulean,T-Sapphire, and alkaline phosphatase. For example, if the first reporterand the second reporter are fluorescent proteins (e.g., CFP and YFP),cells comprising these reporters can be selected by flow cytometry toselect for dual-positive cells. The dual-positive cells can then becombined to generate a polyclonal line, or monoclonal lines can begenerated from single dual-positive cells.

As another example, selection markers can be used to screen for cellsthat have the Cas protein, the first tau repeat domain linked to thefirst reporter, or the second tau repeat domain linked to the secondreporter. Exemplary selection markers include neomycinphosphotransferase (neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), or herpessimplex virus thymidine kinase (HSV-k). Another exemplary selectionmarker is bleomycin resistance protein, encoded by the Sh ble gene(Streptoalloteichus hindustanus bleomycin gene), which confers zeocin(phleomycin D1) resistance.

Aggregation-positive (Agg[+]) cells in which the tau repeat domainstably presents in an aggregated state, meaning that the tau repeatdomain aggregates stably persist in all cells with growth and multiplepassages over time, can be generated, for example, by seeding with tauaggregates. For example, naïve aggregation-negative (Agg[−]) Cas/taubiosensor cells disclosed herein can be treated with recombinantfibrillized tau (e.g., recombinant fibrillized tau repeat domain) toseed the aggregation of the tau repeat domain protein stably expressedby these cells. Likewise, naïve aggregation-negative (Agg[−]) SAM/taubiosensor cells disclosed herein can be treated with recombinantfibrillized tau (e.g., recombinant fibrillized tau repeat domain) toseed the aggregation of the tau repeat domain protein stably expressedby these cells. The fibrillized tau repeat domain can be the same as,similar to, or different from the tau repeat domain stably expressed bythe cells. Optionally, the recombinant fibrillized tau can be mixed withlipofectamine reagent. The seeded cells can then be serially diluted toobtain single-cell-derived clones and to identify clonal cell lines inwhich tau repeat domain aggregates stably persist in all cells withgrowth and multiple passages over time.

As another example, aggregation-positive (Agg[+]) cells in which the taurepeat domain stably presents in an aggregated state, meaning that thetau repeat domain aggregates stably persist in all cells with growth andmultiple passages over time, can be generated, for example, by seedingcells (e.g., tau aggregation-negative cells) with cell lysate from tauaggregation-positive cells. This is the “maximal seeding” described inthe examples herein. For example, the cells can be seeded using a mediumcomprising the cell lysate (e.g., fresh medium comprising the celllysate). “Maximal seeding” can refer to seeding that, by itself, inducestau aggregation in a majority of aggregation-negative tau biosensorcells. “Minimal seeding” can refer to seeding that, by itself, isinsufficient to induce tau aggregation in aggregation-negative taubiosensor cells (or only minimally induces tau aggregation) butsensitizes such cells to induction of aggregation.

The amount or concentration of the cell lysate in the medium can be anysuitable amount or concentration. For example, the concentration of celllysate in the medium can be between about 0.1 μg/mL and about 50 μg/mL,between about 0.1 μg/mL and about 25 μg/mL, between about 0.1 μg/mL andabout 10 μg/mL, between about 0.1 μg/mL and about 5 μg/mL, between about0.1 μg/mL and about 4.5 μg/mL, between about 0.1 μg/mL and about 4μg/mL, between about 0.1 μg/mL and about 3.5 μg/mL, between about 0.1μg/mL and about 3 μg/mL, between about 0.1 μg/mL and about 2.5 μg/mL,between about 0.1 μg/mL and about 2 μg/mL, between about 0.1 μg/mL andabout 1.5 μg/mL, between about 0.1 μg/mL and about 1 μg/mL, betweenabout 0.5 μg/mL and about 50 μg/mL, between about 0.5 μg/mL and about 25μg/mL, between about 0.5 μg/mL and about 10 μg/mL, between about 0.5μg/mL and about 5 μg/mL, between about 0.5 μg/mL and about 4.5 μg/mL,between about 0.5 μg/mL and about 4 μg/mL, between about 0.5 μg/mL andabout 3.5 μg/mL, between about 0.5 μg/mL and about 3 μg/mL, betweenabout 0.5 μg/mL and about 2.5 μg/mL, between about 0.5 μg/mL and about 2μg/mL, between about 0.5 μg/mL and about 1.5 μg/mL, between about 0.5μg/mL and about 1 μg/mL, between about 1 μg/mL and about 50 μg/mL,between about 1 μg/mL and about 25 μg/mL, between about 1 μg/mL andabout 10 μg/mL, between about 1 μg/mL and about 5 μg/mL, between about 1μg/mL and about 4.5 μg/mL, between about 1 μg/mL and about 4 μg/mL,between about 1 μg/mL and about 3.5 μg/mL, between about 1 μg/mL andabout 3 μg/mL, between about 1 μg/mL and about 2.5 μg/mL, between about1 μg/mL and about 2 μg/mL, between about 1 μg/mL and about 1.5 μg/mL,between about 1.5 μg/mL and about 50 μg/mL, between about 1.5 μg/mL andabout 25 μg/mL, between about 1.5 μg/mL and about 10 μg/mL, betweenabout 1.5 μg/mL and about 5 μg/mL, between about 1.5 μg/mL and about 4.5μg/mL, between about 1.5 μg/mL and about 4 μg/mL, between about 1.5μg/mL and about 3.5 μg/mL, between about 1.5 μg/mL and about 3 μg/mL,between about 1.5 μg/mL and about 2.5 μg/mL, between about 1.5 μg/mL andabout 2 μg/mL, between about 2 μg/mL and about 50 μg/mL, between about 2μg/mL and about 25 μg/mL, between about 2 μg/mL and about 10 μg/mL,between about 2 μg/mL and about 5 μg/mL, between about 2 μg/mL and about4.5 μg/mL, between about 2 μg/mL and about 4 μg/mL, between about 2μg/mL and about 3.5 μg/mL, between about 2 μg/mL and about 3 μg/mL,between about 2 μg/mL and about 2.5 μg/mL, between about 2.5 μg/mL andabout 50 μg/mL, between about 2.5 μg/mL and about 25 μg/mL, betweenabout 2.5 μg/mL and about 10 μg/mL, between about 2.5 μg/mL and about 5μg/mL, between about 2.5 μg/mL and about 4.5 μg/mL, between about 2.5μg/mL and about 4 μg/mL, between about 2.5 μg/mL and about 3.5 μg/mL, orbetween about 2.5 μg/mL and about 3 μg/mL of medium (e.g., fresh culturemedium). For example, the cell lysate in the culture medium can be at aconcentration of between about 1 μg/mL and about 5 μg/mL or can be at aconcentration of about 1.5 μg/mL, about 2 μg/mL, about 2.5 μg/mL, about3 μg/mL, about 3.5 μg/mL, about 4 g/mL, about 4.5 μg/mL, or about 5μg/mL. Optionally, the cell lysate can be in a buffer, such asphosphate-buffered saline. Optionally, the buffer can comprise proteaseinhibitors. Examples of protease inhibitors include, but are not limitedto, AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A, andethylenediaminetetracetic acid (EDTA). The buffer can comprise any ofthese inhibitors or any combination thereof (e.g., the buffer cancomprise all of these protease inhibitors).

The cells for producing the lysate can be collected in a buffer, such asphosphate-buffered saline. Optionally, the buffer can comprise proteaseinhibitors. Examples of protease inhibitors include, but are not limitedto, AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A, andethylenediaminetetracetic acid (EDTA). The buffer can comprise any ofthese inhibitors or any combination thereof (e.g., the buffer cancomprise all of these protease inhibitors).

The cell lysate can, for example, be collected by sonicating thetau-aggregation-positive cells (e.g., cells collected in a buffer andprotease inhibitors as described above) for any suitable amount of time.For example, the cells can be sonicated for between about 1 minute andabout 6 minutes, between about 1 minute and about 5 minutes, betweenabout 1 minute and about 4 minutes, between about 1 minute and about 3minutes, between about 2 minutes and about 6 minutes, between about 2minutes and about 5 minutes, between about 2 minutes and about 4minutes, between about 2 minutes and about 3 minutes, between about 2minutes and about 6 minutes, between about 3 minutes and about 5minutes, or between about 3 minutes and about 4 minutes. For example,the cells can be sonicated for between about 2 minutes and about 4minutes or for about 3 minutes.

Optionally, the medium comprises lipofectamine or liposomes (e.g.,cationic liposomes) or phospholipids or another transfection agent.Optionally, the medium comprises lipofectamine. Optionally, the mediumdoes not comprise lipofectamine or liposomes (e.g., cationic liposomes)or phospholipids or another transfection agent. Optionally, the mediumdoes not comprise lipofectamine. The amount or concentration of thelipofectamine or liposomes (e.g., cationic liposomes) or phospholipidsor other transfection agent in the medium can be any suitable amount orconcentration. For example, the concentration of lipofectamine orliposomes (e.g., cationic liposomes) or phospholipids or othertransfection agent in the medium can be between about 0.5 μL/mL to about10 μL/mL, between about 0.5 μL/mL to about 5 μL/mL, between about 0.5μL/mL to about 4.5 μL/mL, between about 0.5 μL/mL to about 4 μL/mL,between about 0.5 μL/mL to about 3.5 μL/mL, between about 0.5 μL/mL toabout 3 μL/mL, between about 0.5 μL/mL to about 2.5 μL/mL, between about0.5 μL/mL to about 2 μL/mL, between about 0.5 μL/mL to about 1.5 μL/mL,between about 0.5 μL/mL to about 1 μL/mL, between about 1 μL/mL to about10 μL/mL, between about 1 μL/mL to about 5 μL/mL, between about 1 μL/mLto about 4.5 μL/mL, between about 1 μL/mL to about 4 μL/mL, betweenabout 1 L/mL to about 3.5 μL/mL, between about 1 μL/mL to about 3 μL/mL,between about 1 μL/mL to about 2.5 μL/mL, between about 1 μL/mL to about2 μL/mL, between about 1 μL/mL to about 1.5 μL/mL, between about 1.5μL/mL to about 10 μL/mL, between about 1.5 μL/mL to about 5 L/mL,between about 1.5 μL/mL to about 4.5 μL/mL, between about 1.5 μL/mL toabout 4 L/mL, between about 1.5 μL/mL to about 3.5 μL/mL, between about1.5 μL/mL to about 3 L/mL, between about 1.5 μL/mL to about 2.5 μL/mL,between about 1.5 μL/mL to about 2 L/mL, between about 2 μL/mL to about10 μL/mL, between about 2 μL/mL to about 5 μL/mL, between about 2 μL/mLto about 4.5 μL/mL, between about 2 μL/mL to about 4 μL/mL, betweenabout 2 μL/mL to about 3.5 μL/mL, between about 2 μL/mL to about 3μL/mL, or between about 2 μL/mL to about 2.5 μL/mL of medium (e.g.,fresh medium). For example, the concentration of lipofectamine orliposomes (e.g., cationic liposomes) or phospholipids or othertransfection agent in the medium can be between about 1.5 μL/mL andabout 4 μL/mL or it can be about 1.5 L/mL, about 2 μL/mL, about 2.5μL/mL, about 3 μL/mL, about 3.5 μL/mL, or about 4 μL/mL.

Tau cell-to-cell propagation may also result from tau aggregationactivity secreted by aggregate-containing cells. For example, Agg[+]cells, or cells sensitized to becoming Agg[+] cells (e.g., sensitized totau seeding or tau aggregation activity), can be generated byco-culturing Agg[−] Cas/tau biosensor cells with Agg[+] cells. Likewise,Agg[+] cells, or cells sensitized to becoming Agg[+] cells (e.g.,sensitized to tau seeding or tau aggregation activity), can be generatedby co-culturing Agg[−] SAM/tau biosensor cells with Agg[+] cells.

Agg[+] cells, or cells sensitized to becoming Agg[+] cells (e.g.,sensitized to tau seeding or tau aggregation activity), can also begenerated using conditioned medium harvested from culturedtau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state as described herein. This is the“minimal seeding” disclosed in the examples herein. Conditioned mediumrefers to spent medium harvested from cultured cells. It containsmetabolites, growth factors, and extracellular matrix proteins secretedinto the medium by the cultured cells. Use of conditioned medium doesnot involve co-culturing with Agg[+] cells (i.e., the naïve Agg[−] cellsare not co-cultured with Agg[+] cells). As one example, conditionedmedium can be generated by collecting medium that has been on confluentAgg[+] cells. The medium can have been on the confluent Agg[+] cells forabout 12 hours, about 24 hours, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, or about 10 days. For example, the medium can have been on theconfluent Agg[+] cells for about 1 to about 7, about 2 to about 6, about3 to about 5, or about 4 days. Conditioned medium can then be applied tonaïve (Agg[−]) Cas/tau biosensor cells in combination with fresh medium.Likewise, conditioned medium can then be applied to naïve (Agg[−])SAM/tau biosensor cells in combination with fresh medium. The ratio ofconditioned medium to fresh medium can be, for example, about 10:1,about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. For example,the ratio of conditioned medium of fresh medium can be from about 5:1 toabout 1:1, about 4:1 to about 2:1, or about 3:1. For example, it cancomprise culturing the genetically modified population of cells in about90% conditioned medium and about 10% fresh medium, about 85% conditionedmedium and about 15% fresh medium, about 80% conditioned medium andabout 20% fresh medium, about 75% conditioned medium and about 25% freshmedium, about 70% conditioned medium and about 30% fresh medium, about65% conditioned medium and about 35% fresh medium, about 60% conditionedmedium and about 40% fresh medium, about 55% conditioned medium andabout 45% fresh medium, about 50% conditioned medium and about 50% freshmedium, about 45% conditioned medium and about 55% fresh medium, about40% conditioned medium and about 60% fresh medium, about 35% conditionedmedium and about 65% fresh medium, about 30% conditioned medium andabout 70% fresh medium, about 25% conditioned medium and about 75% freshmedium, about 20% conditioned medium and about 80% fresh medium, about15% conditioned medium and about 85% fresh medium, or about 10%conditioned medium and about 90% fresh medium. In one example, it cancomprise culturing the genetically modified population of cells in amedium that comprises at least about 50% conditioned medium and no morethan about 50% fresh medium. In a specific example, it can compriseculturing the genetically modified population of cells in about 75%conditioned medium and about 25% fresh medium. Optionally, theconditioned medium is applied to the naïve Agg[−] cells withoutlipofectamine or without liposomes (e.g., cationic liposomes) or withoutphospholipids. Optionally, the genetically modified population of cellsis not co-cultured with the tau-aggregation-positive cells in which atau repeat domain stably presents in an aggregated state.

Conditioned medium without co-culturing has not been used in thiscontext as a seeding agent before. However, conditioned medium isparticularly useful for large-scale genome-wide screens because taufibrils produced in vitro are a limited resource. In addition,conditioned medium is more physiologically relevant because it isproduced and secreted by cells rather than in vitro. Use of conditionedmedium as described herein provides a boost of tau seeding activity(e.g., ˜0.1% as measured by FRET induction as disclosed elsewhereherein) to sensitize cells to tau aggregation.

C. In Vitro Cultures and Conditioned Medium

Also disclosed herein are in vitro cultures or compositions comprisingthe Cas/tau biosensor cells disclosed herein and medium for culturingthose cells. Also disclosed herein are in vitro cultures or compositionscomprising the SAM/tau biosensor cells disclosed herein and medium forculturing those cells. The cells can be Agg[−] cells or Agg[+] cells.For example, the culture or composition can comprise Agg[−] cells. Inone example, the medium comprises conditioned medium from Agg[+] cellsas disclosed elsewhere herein. Optionally, the cells in the culture orcomposition are Agg[−] cells and are not being co-cultured with Agg[+]cells. The medium can comprise a mixture of conditioned medium and freshmedium. As one example, the ratio of conditioned medium to fresh mediumcan be, for example, about 10:1, about 9:1, about 8:1, about 7:1, about6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2,about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about1:9, or about 1:10. For example, the ratio of conditioned medium offresh medium can be from about 5:1 to about 1:1, about 4:1 to about 2:1,or about 3:1. For example, it can comprise culturing the geneticallymodified population of cells in about 90% conditioned medium and about10% fresh medium, about 85% conditioned medium and about 15% freshmedium, about 80% conditioned medium and about 20% fresh medium, about75% conditioned medium and about 25% fresh medium, about 70% conditionedmedium and about 30% fresh medium, about 65% conditioned medium andabout 35% fresh medium, about 60% conditioned medium and about 40% freshmedium, about 55% conditioned medium and about 45% fresh medium, about50% conditioned medium and about 50% fresh medium, about 45% conditionedmedium and about 55% fresh medium, about 40% conditioned medium andabout 60% fresh medium, about 35% conditioned medium and about 65% freshmedium, about 30% conditioned medium and about 70% fresh medium, about25% conditioned medium and about 75% fresh medium, about 20% conditionedmedium and about 80% fresh medium, about 15% conditioned medium andabout 85% fresh medium, or about 10% conditioned medium and about 90%fresh medium. In one example, it can comprise culturing the geneticallymodified population of cells in a medium that comprises at least about50% conditioned medium and no more than about 50% fresh medium. In aspecific example, it can comprise culturing the genetically modifiedpopulation of cells in about 75% conditioned medium and about 25% freshmedium. Optionally, the medium comprises lipofectamine or liposomes(e.g., cationic liposomes) or phospholipids. Optionally, the medium doesnot comprise lipofectamine or liposomes (e.g., cationic liposomes) orphospholipids. Optionally, the medium does not comprise lipofectamine.

D. In Vitro Cultures and Medium Comprising Lysate fromTau-Aggregation-Positive Cells

Also disclosed herein are in vitro cultures or compositions comprisingthe Cas/tau biosensor cells disclosed herein and medium for culturingthose cells. Also disclosed herein are in vitro cultures or compositionscomprising the SAM/tau biosensor cells disclosed herein and medium forculturing those cells. In one example, the medium comprises a celllysate from cultured tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state. The cells can beAgg[−] cells or Agg[+] cells. For example, the culture or compositioncan comprise Agg[−] cells. Optionally, the cells in the culture orcomposition are Agg[−] cells and are not being co-cultured with Agg[+]cells. The medium can comprise a mixture of fresh medium and the celllysate.

The amount or concentration of the cell lysate in the medium can be anysuitable amount or concentration. For example, the concentration of celllysate in the medium can be between about 0.1 μg/mL and about 50 μg/mL,between about 0.1 μg/mL and about 25 μg/mL, between about 0.1 μg/mL andabout 10 μg/mL, between about 0.1 μg/mL and about 5 μg/mL, between about0.1 μg/mL and about 4.5 μg/mL, between about 0.1 μg/mL and about 4μg/mL, between about 0.1 μg/mL and about 3.5 μg/mL, between about 0.1μg/mL and about 3 μg/mL, between about 0.1 μg/mL and about 2.5 μg/mL,between about 0.1 μg/mL and about 2 μg/mL, between about 0.1 μg/mL andabout 1.5 μg/mL, between about 0.1 μg/mL and about 1 μg/mL, betweenabout 0.5 μg/mL and about 50 μg/mL, between about 0.5 μg/mL and about 25μg/mL, between about 0.5 μg/mL and about 10 μg/mL, between about 0.5μg/mL and about 5 μg/mL, between about 0.5 μg/mL and about 4.5 μg/mL,between about 0.5 μg/mL and about 4 μg/mL, between about 0.5 μg/mL andabout 3.5 μg/mL, between about 0.5 μg/mL and about 3 μg/mL, betweenabout 0.5 μg/mL and about 2.5 μg/mL, between about 0.5 μg/mL and about 2μg/mL, between about 0.5 μg/mL and about 1.5 μg/mL, between about 0.5μg/mL and about 1 μg/mL, between about 1 μg/mL and about 50 μg/mL,between about 1 μg/mL and about 25 μg/mL, between about 1 μg/mL andabout 10 μg/mL, between about 1 μg/mL and about 5 μg/mL, between about 1μg/mL and about 4.5 μg/mL, between about 1 μg/mL and about 4 μg/mL,between about 1 μg/mL and about 3.5 μg/mL, between about 1 μg/mL andabout 3 μg/mL, between about 1 μg/mL and about 2.5 μg/mL, between about1 μg/mL and about 2 μg/mL, between about 1 μg/mL and about 1.5 μg/mL,between about 1.5 μg/mL and about 50 μg/mL, between about 1.5 μg/mL andabout 25 μg/mL, between about 1.5 μg/mL and about 10 μg/mL, betweenabout 1.5 μg/mL and about 5 μg/mL, between about 1.5 μg/mL and about 4.5μg/mL, between about 1.5 μg/mL and about 4 μg/mL, between about 1.5μg/mL and about 3.5 μg/mL, between about 1.5 μg/mL and about 3 μg/mL,between about 1.5 μg/mL and about 2.5 μg/mL, between about 1.5 μg/mL andabout 2 μg/mL, between about 2 μg/mL and about 50 μg/mL, between about 2μg/mL and about 25 μg/mL, between about 2 μg/mL and about 10 μg/mL,between about 2 μg/mL and about 5 μg/mL, between about 2 μg/mL and about4.5 μg/mL, between about 2 μg/mL and about 4 μg/mL, between about 2μg/mL and about 3.5 μg/mL, between about 2 μg/mL and about 3 μg/mL,between about 2 μg/mL and about 2.5 μg/mL, between about 2.5 μg/mL andabout 50 μg/mL, between about 2.5 μg/mL and about 25 μg/mL, betweenabout 2.5 μg/mL and about 10 μg/mL, between about 2.5 μg/mL and about 5μg/mL, between about 2.5 μg/mL and about 4.5 μg/mL, between about 2.5μg/mL and about 4 μg/mL, between about 2.5 μg/mL and about 3.5 μg/mL, orbetween about 2.5 μg/mL and about 3 μg/mL of medium (e.g., fresh culturemedium). For example, the cell lysate in the culture medium can be at aconcentration of between about 1 μg/mL and about 5 μg/mL or can be at aconcentration of about 1.5 μg/mL, about 2 μg/mL, about 2.5 μg/mL, about3 μg/mL, about 3.5 μg/mL, about 4 g/mL, about 4.5 μg/mL, or about 5μg/mL. Optionally, the cell lysate can be in a buffer, such asphosphate-buffered saline. Optionally, the buffer can comprise proteaseinhibitors. Examples of protease inhibitors include, but are not limitedto, AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A, andethylenediaminetetracetic acid (EDTA). The buffer can comprise any ofthese inhibitors or any combination thereof (e.g., the buffer cancomprise all of these protease inhibitors).

The cells for producing the lysate can be collected in a buffer, such asphosphate-buffered saline. Optionally, the buffer can comprise proteaseinhibitors. Examples of protease inhibitors include, but are not limitedto, AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A, andethylenediaminetetracetic acid (EDTA). The buffer can comprise any ofthese inhibitors or any combination thereof (e.g., the buffer cancomprise all of these protease inhibitors).

The cell lysate can, for example, be collected by sonicating thetau-aggregation-positive cells (e.g., cells collected in a buffer andprotease inhibitors as described above) for any suitable amount of time.For example, the cells can be sonicated for between about 1 minute andabout 6 minutes, between about 1 minute and about 5 minutes, betweenabout 1 minute and about 4 minutes, between about 1 minute and about 3minutes, between about 2 minutes and about 6 minutes, between about 2minutes and about 5 minutes, between about 2 minutes and about 4minutes, between about 2 minutes and about 3 minutes, between about 2minutes and about 6 minutes, between about 3 minutes and about 5minutes, or between about 3 minutes and about 4 minutes. For example,the cells can be sonicated for between about 2 minutes and about 4minutes or for about 3 minutes.

Optionally, the medium comprises lipofectamine or liposomes (e.g.,cationic liposomes) or phospholipids or another transfection agent.Optionally, the medium comprises lipofectamine. Optionally, the mediumdoes not comprise lipofectamine or liposomes (e.g., cationic liposomes)or phospholipids or another transfection agent. Optionally, the mediumdoes not comprise lipofectamine. The amount or concentration of thelipofectamine or liposomes (e.g., cationic liposomes) or phospholipidsor other transfection agent in the medium can be any suitable amount orconcentration. For example, the concentration of lipofectamine orliposomes (e.g., cationic liposomes) or phospholipids or othertransfection agent in the medium can be between about 0.5 μL/mL to about10 μL/mL, between about 0.5 μL/mL to about 5 μL/mL, between about 0.5μL/mL to about 4.5 μL/mL, between about 0.5 μL/mL to about 4 μL/mL,between about 0.5 μL/mL to about 3.5 μL/mL, between about 0.5 μL/mL toabout 3 μL/mL, between about 0.5 μL/mL to about 2.5 μL/mL, between about0.5 μL/mL to about 2 μL/mL, between about 0.5 μL/mL to about 1.5 μL/mL,between about 0.5 μL/mL to about 1 μL/mL, between about 1 μL/mL to about10 μL/mL, between about 1 μL/mL to about 5 μL/mL, between about 1 μL/mLto about 4.5 μL/mL, between about 1 μL/mL to about 4 μL/mL, betweenabout 1 L/mL to about 3.5 μL/mL, between about 1 μL/mL to about 3 μL/mL,between about 1 μL/mL to about 2.5 μL/mL, between about 1 μL/mL to about2 μL/mL, between about 1 μL/mL to about 1.5 μL/mL, between about 1.5μL/mL to about 10 μL/mL, between about 1.5 μL/mL to about 5 L/mL,between about 1.5 μL/mL to about 4.5 μL/mL, between about 1.5 μL/mL toabout 4 L/mL, between about 1.5 μL/mL to about 3.5 μL/mL, between about1.5 μL/mL to about 3 L/mL, between about 1.5 μL/mL to about 2.5 μL/mL,between about 1.5 μL/mL to about 2 L/mL, between about 2 μL/mL to about10 μL/mL, between about 2 μL/mL to about 5 μL/mL, between about 2 μL/mLto about 4.5 μL/mL, between about 2 μL/mL to about 4 μL/mL, betweenabout 2 μL/mL to about 3.5 μL/mL, between about 2 μL/mL to about 3μL/mL, or between about 2 μL/mL to about 2.5 μL/mL of medium (e.g.,fresh medium). For example, the concentration of lipofectamine orliposomes (e.g., cationic liposomes) or phospholipids or othertransfection agent in the medium can be between about 1.5 μL/mL andabout 4 μL/mL or it can be about 1.5 L/mL, about 2 μL/mL, about 2.5μL/mL, about 3 μL/mL, about 3.5 μL/mL, or about 4 μL/mL.

III. Guide RNA Knockout Libraries

The CRISPRn screening methods disclosed herein make use of CRISPR guideRNA (gRNA) knockout libraries such as genome-wide gRNA knockoutlibraries. Cas nucleases such as Cas9 can be programmed to inducedouble-strand breaks at specific genomic loci through gRNAs designed totarget specific target sequences. Because the targeting specificity ofCas proteins is conferred by short gRNAs, pooled genome-scale functionalscreening is possible. Such libraries have several advantages overlibraries such as shRNA libraries, which reduce protein expression bytargeting mRNA. In contrast, gRNA libraries achieve knockout viaframeshift mutations introduced in genomic coding regions of genes.

The CRISPRa screening methods disclosed herein make use of CRISPR guideRNA (gRNA) transcriptional activation libraries such as genome-wide gRNAtranscriptional activation libraries. SAM systems can be programmed toactivate transcription of genes at specific genomic loci through gRNAsdesigned to target specific target sequences. Because the targetingspecificity of Cas proteins is conferred by short gRNAs, pooledgenome-scale functional screening is possible.

The gRNAs in a library can target any number of genes. For example, thegRNAs can target about 50 or more genes, about 100 or more genes, about200 or more genes, about 300 or more genes, about 400 or more genes,about 500 or more genes, about 1000 or more genes, about 2000 or moregenes, about 3000 or more genes, about 4000 or more genes, about 5000 ormore genes, about 10000 or more genes, or about 20000 or more genes. Insome libraries, the gRNAs can be selected to target genes in aparticular signaling pathway. Some libraries are genome-wide libraries.

The genome-wide libraries include one or more gRNAs (e.g., sgRNAs)targeting each gene in the genome. The genome being targeted can anytype of genome. For example, the genome can be a eukaryotic genome, amammalian genome, a non-human mammalian genome, a rodent genome, a mousegenome, a rat genome, or a human genome. In one example, the targetedgenome is a human genome.

The gRNAs can target any number of sequences within each individualtargeted gene. In some libraries, a plurality of target sequences aretargeted on average in each of the targeted plurality of genes. Forexample, about 2 to about 10, about 2 to about 9, about 2 to about 8,about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 toabout 4, or about 2 to about 3 unique target sequences can be targetedon average in each of the targeted plurality of genes. For example, atleast about 2, at least about 3, at least about 4, at least about 5, orat least about 6 unique target sequences can be targeted on average ineach of the targeted plurality of genes. As a specific example, about 6target sequences can be targeted on average in each of the targetedplurality of genes. As another specific example, about 3 to about 6 orabout 4 to about 6 target sequences are targeted on average in each ofthe targeted plurality of genes.

For example, the libraries can target genes with an average coverage ofabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, or about 10 gRNAs per gene. In a specific example a library cantarget genes with an average coverage of about 3-4 gRNAs per gene orabout 6 gRNAs per gene.

The gRNAs can target any desired location in the target genes. TheCRISPRn gRNAs can be designed to target coding regions of genes so thatcleavage by the corresponding Cas protein will result in frameshiftinsertion/deletion (indel) mutations that result in a loss-of-functionallele. More specifically, frameshift mutations can be achieved throughtargeted DNA double strand breaks and subsequent mutagenic repair viathe non-homologous end joining (NHEJ) pathway, which produces indels atthe site of break. The indel being introduced into the DSB is random,with some indels leading to frameshift mutations that cause prematuretermination of the gene.

In some CRISPRn libraries, each gRNA targets a constitutive exon ifpossible. In some CRISPRn libraries, each gRNA targets a 5′ constitutiveexon if possible. In some methods, each gRNA targets a first exon, asecond exon, or a third exon (from the 5′ end of the gene) if possible.

As one example, the gRNAs in the CRISPRn library can target constitutiveexons. Constitutive exons are exons that are consistently conservedafter splicing. Exons expressed across all tissues can be consideredconstitutive exons for gRNA targeting. The gRNAs in the library cantarget constitutive exons near the 5′ end of each gene. Optionally, thefirst and last exons of each gene can be excluded as potential targets.Optionally, any exon containing an alternative splicing site can beexcluded as potential targets. Optionally, the two earliest exonsmeeting the above criteria are selected as potential targets.Optionally, exons 2 and 3 are selected as potential targets (e.g., if noconstitutive exons are identified). In addition, the gRNAs in thelibrary can be selected and designed to minimize off-target effects.

In a specific example, the genome-wide CRISPRn gRNA library or librariescomprise sgRNAs targeting 5′ constitutive exons of >18,000 genes in thehuman genome with an average coverage of ˜6 sgRNAs per gene, with eachtarget site was selected to minimize off-target modification.

The CRISPRa gRNAs can be designed to target sequences adjacent to thetranscription start site of a gene. For example, the target sequence canbe within 1000, 900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10,5, or 1 base pair of the transcription start site. For example, eachgRNA in the CRISPRa library can target a sequence within 200 bp upstreamof a transcription start site. Optionally, the target sequence is withinthe region 200 base pairs upstream of the transcription start site and 1base pair downstream of the transcription start site (−200 to +1).

The gRNAs in the genome-wide library can be in any form. For example,the gRNA library can be packaged in a viral vector, such as retroviralvectors, lentiviral vectors, or adenoviral vectors. In a specificexample, the gRNA library is packaged in lentiviral vectors. The vectorscan further comprise reporter genes or selection markers to facilitateselection of cells that receive the vectors. Examples of such reportergenes and selection markers are disclosed elsewhere herein. As oneexample, the selection marker can be one that imparts resistance to adrug, such as neomycin phosphotransferase, hygromycin Bphosphotransferase, puromycin-N-acetyltransferase, and blasticidin Sdeaminase. Another exemplary selection marker is bleomycin resistanceprotein, encoded by the Sh ble gene (Streptoalloteichus hindustanusbleomycin gene), which confers zeocin (phleomycin D1) resistance. Forexample, cells can be selected with a drug (e.g., puromycin) so thatonly cells transduced with a guide RNA construct are preserved for beingused to carry out screening.

A. Guide RNAs

A “guide RNA” or “gRNA” is an RNA molecule that binds to a Cas protein(e.g., Cas9 protein) and targets the Cas protein to a specific locationwithin a target DNA. Guide RNAs can comprise two segments: a“DNA-targeting segment” and a “protein-binding segment.” “Segment”includes a section or region of a molecule, such as a contiguous stretchof nucleotides in an RNA. Some gRNAs, such as those for Cas9, cancomprise two separate RNA molecules: an “activator-RNA” (e.g., tracrRNA)and a “targeter-RNA” (e.g., CRISPR RNA or crRNA). Other gRNAs are asingle RNA molecule (single RNA polynucleotide), which can also becalled a “single-molecule gRNA,” a “single-guide RNA,” or an “sgRNA.”See, e.g., WO 2013/176772, WO 2014/065596, WO 2014/089290, WO2014/093622, WO 2014/099750, WO 2013/142578, and WO 2014/131833, each ofwhich is herein incorporated by reference in its entirety for allpurposes. For Cas9, for example, a single-guide RNA can comprise a crRNAfused to a tracrRNA (e.g., via a linker). For Cpf1, for example, only acrRNA is needed to achieve binding to a target sequence. The terms“guide RNA” and “gRNA” include both double-molecule (i.e., modular)gRNAs and single-molecule gRNAs.

An exemplary two-molecule gRNA comprises a crRNA-like (“CRISPR RNA” or“targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and acorresponding tracrRNA-like (“trans-acting CRISPR RNA” or“activator-RNA” or “tracrRNA”) molecule. A crRNA comprises both theDNA-targeting segment (single-stranded) of the gRNA and a stretch ofnucleotides that forms one half of the dsRNA duplex of theprotein-binding segment of the gRNA. An example of a crRNA tail, locateddownstream (3′) of the DNA-targeting segment, comprises, consistsessentially of, or consists of GUUUUAGAGCUAUGCU (SEQ ID NO: 23). Any ofthe DNA-targeting segments disclosed herein can be joined to the 5′ endof SEQ ID NO: 23 to form a crRNA.

A corresponding tracrRNA (activator-RNA) comprises a stretch ofnucleotides that forms the other half of the dsRNA duplex of theprotein-binding segment of the gRNA. A stretch of nucleotides of a crRNAare complementary to and hybridize with a stretch of nucleotides of atracrRNA to form the dsRNA duplex of the protein-binding domain of thegRNA. As such, each crRNA can be said to have a corresponding tracrRNA.An example of a tracrRNA sequence comprises, consists essentially of, orconsists of AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU (SEQ ID NO: 24). Other examples of tracrRNA sequencescomprise, consist essentially of, or consist of any one of

(SEQ ID NO: 28) AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU, or (SEQ ID NO: 29)GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

In systems in which both a crRNA and a tracrRNA are needed, the crRNAand the corresponding tracrRNA hybridize to form a gRNA. In systems inwhich only a crRNA is needed, the crRNA can be the gRNA. The crRNAadditionally provides the single-stranded DNA-targeting segment thathybridizes to the complementary strand of a target DNA. If used formodification within a cell, the exact sequence of a given crRNA ortracrRNA molecule can be designed to be specific to the species in whichthe RNA molecules will be used. See, e.g., Mali et al. (2013) Science339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al.(2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol.31:233-239; and Cong et al. (2013) Science 339:819-823, each of which isherein incorporated by reference in its entirety for all purposes.

The DNA-targeting segment (crRNA) of a given gRNA comprises a nucleotidesequence that is complementary to a sequence on the complementary strandof the target DNA, as described in more detail below. The DNA-targetingsegment of a gRNA interacts with the target DNA in a sequence-specificmanner via hybridization (i.e., base pairing). As such, the nucleotidesequence of the DNA-targeting segment may vary and determines thelocation within the target DNA with which the gRNA and the target DNAwill interact. The DNA-targeting segment of a subject gRNA can bemodified to hybridize to any desired sequence within a target DNA.Naturally occurring crRNAs differ depending on the CRISPR/Cas system andorganism but often contain a targeting segment of between 21 to 72nucleotides length, flanked by two direct repeats (DR) of a length ofbetween 21 to 46 nucleotides (see, e.g., WO 2014/131833, hereinincorporated by reference in its entirety for all purposes). In the caseof S. pyogenes, the DRs are 36 nucleotides long and the targetingsegment is 30 nucleotides long. The 3′ located DR is complementary toand hybridizes with the corresponding tracrRNA, which in turn binds tothe Cas protein.

The DNA-targeting segment can have, for example, a length of at leastabout 12, 15, 17, 18, 19, 20, 25, 30, 35, or 40 nucleotides. SuchDNA-targeting segments can have, for example, a length from about 12 toabout 100, from about 12 to about 80, from about 12 to about 50, fromabout 12 to about 40, from about 12 to about 30, from about 12 to about25, or from about 12 to about 20 nucleotides. For example, the DNAtargeting segment can be from about 15 to about 25 nucleotides (e.g.,from about 17 to about 20 nucleotides, or about 17, 18, 19, or 20nucleotides). See, e.g., US 2016/0024523, herein incorporated byreference in its entirety for all purposes. For Cas9 from S. pyogenes, atypical DNA-targeting segment is between 16 and 20 nucleotides in lengthor between 17 and 20 nucleotides in length. For Cas9 from S. aureus, atypical DNA-targeting segment is between 21 and 23 nucleotides inlength. For Cpf1, a typical DNA-targeting segment is at least 16nucleotides in length or at least 18 nucleotides in length.

TracrRNAs can be in any form (e.g., full-length tracrRNAs or activepartial tracrRNAs) and of varying lengths. They can include primarytranscripts or processed forms. For example, tracrRNAs (as part of asingle-guide RNA or as a separate molecule as part of a two-moleculegRNA) may comprise, consist essentially of, or consist of all or aportion of a wild type tracrRNA sequence (e.g., about or more than about20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild typetracrRNA sequence). Examples of wild type tracrRNA sequences from S.pyogenes include 171-nucleotide, 89-nucleotide, 75-nucleotide, and65-nucleotide versions. See, e.g., Deltcheva et al. (2011) Nature471:602-607; WO 2014/093661, each of which is herein incorporated byreference in its entirety for all purposes. Examples of tracrRNAs withinsingle-guide RNAs (sgRNAs) include the tracrRNA segments found within+48, +54, +67, and +85 versions of sgRNAs, where “+n” indicates that upto the +n nucleotide of wild type tracrRNA is included in the sgRNA. SeeU.S. Pat. No. 8,697,359, herein incorporated by reference in itsentirety for all purposes.

The percent complementarity between the DNA-targeting segment of theguide RNA and the complementary strand of the target DNA can be at least60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, or 100%). The percent complementarity between theDNA-targeting segment and the complementary strand of the target DNA canbe at least 60% over about 20 contiguous nucleotides. As an example, thepercent complementarity between the DNA-targeting segment and thecomplementary strand of the target DNA can be 100% over the 14contiguous nucleotides at the 5′ end of the complementary strand of thetarget DNA and as low as 0% over the remainder. In such a case, theDNA-targeting segment can be considered to be 14 nucleotides in length.As another example, the percent complementarity between theDNA-targeting segment and the complementary strand of the target DNA canbe 100% over the seven contiguous nucleotides at the 5′ end of thecomplementary strand of the target DNA and as low as 0% over theremainder. In such a case, the DNA-targeting segment can be consideredto be 7 nucleotides in length. In some guide RNAs, at least 17nucleotides within the DNA-targeting segment are complementary to thecomplementary strand of the target DNA. For example, the DNA-targetingsegment can be 20 nucleotides in length and can comprise 1, 2, or 3mismatches with the complementary strand of the target DNA. In oneexample, the mismatches are not adjacent to the region of thecomplementary strand corresponding to the protospacer adjacent motif(PAM) sequence (i.e., the reverse complement of the PAM sequence) (e.g.,the mismatches are in the 5′ end of the DNA-targeting segment of theguide RNA, or the mismatches are at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the region ofthe complementary strand corresponding to the PAM sequence).

The protein-binding segment of a gRNA can comprise two stretches ofnucleotides that are complementary to one another. The complementarynucleotides of the protein-binding segment hybridize to form adouble-stranded RNA duplex (dsRNA). The protein-binding segment of asubject gRNA interacts with a Cas protein, and the gRNA directs thebound Cas protein to a specific nucleotide sequence within target DNAvia the DNA-targeting segment.

Single-guide RNAs can comprise a DNA-targeting segment and a scaffoldsequence (i.e., the protein-binding or Cas-binding sequence of the guideRNA). For example, such guide RNAs can have a 5′ DNA-targeting segmentjoined to a 3′ scaffold sequence. Exemplary scaffold sequences comprise,consist essentially of, or consist of:GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (version 1; SEQ ID NO: 17);GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 2; SEQ ID NO: 18);GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 3; SEQ ID NO: 19); andGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (version 4; SEQ ID NO: 20). Otherexemplary scaffold sequences comprise, consist essentially of, orconsist of:

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (version 5; SEQ ID NO: 30);GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (version 6; SEQ ID NO: 31); orGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU  (version 7; SEQ ID NO: 32).Guide RNAs targeting any of the guide RNA target sequences disclosedherein can include, for example, a DNA-targeting segment on the 5′ endof the guide RNA fused to any of the exemplary guide RNA scaffoldsequences on the 3′ end of the guide RNA. That is, any of theDNA-targeting segments disclosed herein can be joined to the 5′ end ofany one of the above scaffold sequences to form a single guide RNA(chimeric guide RNA).

Guide RNAs can include modifications or sequences that provide foradditional desirable features (e.g., modified or regulated stability;subcellular targeting; tracking with a fluorescent label; a binding sitefor a protein or protein complex; and the like). Examples of suchmodifications include, for example, a 5′ cap (e.g., a 7-methylguanylatecap (m7G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); ariboswitch sequence (e.g., to allow for regulated stability and/orregulated accessibility by proteins and/or protein complexes); astability control sequence; a sequence that forms a dsRNA duplex (i.e.,a hairpin); a modification or sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, chloroplasts, and thelike); a modification or sequence that provides for tracking (e.g.,direct conjugation to a fluorescent molecule, conjugation to a moietythat facilitates fluorescent detection, a sequence that allows forfluorescent detection, and so forth); a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like); and combinations thereof. Otherexamples of modifications include engineered stem loop duplexstructures, engineered bulge regions, engineered hairpins 3′ of the stemloop duplex structure, or any combination thereof. See, e.g., US2015/0376586, herein incorporated by reference in its entirety for allpurposes. A bulge can be an unpaired region of nucleotides within theduplex made up of the crRNA-like region and the minimum tracrRNA-likeregion. A bulge can comprise, on one side of the duplex, an unpaired5′-XXXY-3′ where X is any purine and Y can be a nucleotide that can forma wobble pair with a nucleotide on the opposite strand, and an unpairednucleotide region on the other side of the duplex.

In some cases, a transcriptional activation system can be usedcomprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1. GuideRNAs in such systems can be designed with aptamer sequences appended tosgRNA tetraloop and stem-loop 2 designed to bind dimerized MS2bacteriophage coat proteins. See, e.g., Konermann et al. (2015) Nature517(7536):583-588, herein incorporated by reference in its entirety forall purposes.

Unmodified nucleic acids can be prone to degradation. Exogenous nucleicacids can also induce an innate immune response. Modifications can helpintroduce stability and reduce immunogenicity. Guide RNAs can comprisemodified nucleosides and modified nucleotides including, for example,one or more of the following: (1) alteration or replacement of one orboth of the non-linking phosphate oxygens and/or of one or more of thelinking phosphate oxygens in the phosphodiester backbone linkage; (2)alteration or replacement of a constituent of the ribose sugar such asalteration or replacement of the 2′ hydroxyl on the ribose sugar; (3)replacement of the phosphate moiety with dephospho linkers; (4)modification or replacement of a naturally occurring nucleobase; (5)replacement or modification of the ribose-phosphate backbone; (6)modification of the 3′ end or 5′ end of the oligonucleotide (e.g.,removal, modification or replacement of a terminal phosphate group orconjugation of a moiety); and (7) modification of the sugar. Otherpossible guide RNA modifications include modifications of or replacementof uracils or poly-uracil tracts. See, e.g., WO 2015/048577 and US2016/0237455, each of which is herein incorporated by reference in itsentirety for all purposes. Similar modifications can be made toCas-encoding nucleic acids, such as Cas mRNAs. For example, Cas mRNAscan be modified by depletion of uridine using synonymous codons.

As one example, nucleotides at the 5′ or 3′ end of a guide RNA caninclude phosphorothioate linkages (e.g., the bases can have a modifiedphosphate group that is a phosphorothioate group). For example, a guideRNA can include phosphorothioate linkages between the 2, 3, or 4terminal nucleotides at the 5′ or 3′ end of the guide RNA. As anotherexample, nucleotides at the 5′ and/or 3′ end of a guide RNA can have2′-O-methyl modifications. For example, a guide RNA can include2′-O-methyl modifications at the 2, 3, or 4 terminal nucleotides at the5′ and/or 3′ end of the guide RNA (e.g., the 5′ end). See, e.g., WO2017/173054 A1 and Finn et al. (2018) Cell Reports 22:1-9, each of whichis herein incorporated by reference in its entirety for all purposes.Other possible modifications are described in more detail elsewhereherein. In a specific example, a guide RNA includes 2′-O-methyl analogsand 3′ phosphorothioate internucleotide linkages at the first three 5′and 3′ terminal RNA residues. Such chemical modifications can, forexample, provide greater stability and protection from exonucleases toguide RNAs, allowing them to persist within cells for longer thanunmodified guide RNAs. Such chemical modifications can also, forexample, protect against innate intracellular immune responses that canactively degrade RNA or trigger immune cascades that lead to cell death.

In some guide RNAs (e.g., single guide RNAs), at least one loop (e.g.,two loops) of the guide RNA is modified by insertion of a distinct RNAsequence that binds to one or more adaptors (i.e., adaptor proteins ordomains). Such adaptor proteins can be used to further recruit one ormore heterologous functional domains, such as transcriptional activationdomains (e.g., for use in CRISPRa screening in the SAM/tau biosensorcells). Examples of fusion proteins comprising such adaptor proteins(i.e., chimeric adaptor proteins) are disclosed elsewhere herein. Forexample, an MS2-binding loop ggccAACAUGAGGAUCACCCAUGUCUGCAGggcc (SEQ IDNO: 33) may replace nucleotides +13 to +16 and nucleotides +53 to +56 ofthe sgRNA scaffold (backbone) set forth in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) or the sgRNA backbone for the S. pyogenesCRISPR/Cas9 system described in WO 2016/049258 and Konermann et al.(2015) Nature 517(7536):583-588, each of which is herein incorporated byreference in its entirety for all purposes. See also US 2019-0284572 andWO 2019/183123, each of which is herein incorporated by reference in itsentirety for all purposes. The guide RNA numbering used herein refers tothe nucleotide numbering in the guide RNA scaffold sequence (i.e., thesequence downstream of the DNA-targeting segment of the guide RNA). Forexample, the first nucleotide of the guide RNA scaffold is +1, thesecond nucleotide of the scaffold is +2, and so forth. Residuescorresponding with nucleotides +13 to +16 in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) are the loop sequence in the region spanningnucleotides +9 to +21 in SEQ ID NO: 17 or SEQ ID NO: 19 (or SEQ ID NO:30 or 31), a region referred to herein as the tetraloop. Residuescorresponding with nucleotides +53 to +56 in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) are the loop sequence in the region spanningnucleotides +48 to +61 in SEQ ID NO: 17 or SEQ ID NO: 19 (or SEQ ID NO:30 or 31), a region referred to herein as the stem loop 2. Other stemloop sequences in SEQ ID NO: 17 or SEQ ID NO: 19 (or SEQ ID NO: 30 or31) comprise stem loop 1 (nucleotides +33 to +41) and stem loop 3(nucleotides +63 to +75). The resulting structure is an sgRNA scaffoldin which each of the tetraloop and stem loop 2 sequences have beenreplaced by an MS2 binding loop. The tetraloop and stem loop 2 protrudefrom the Cas9 protein in such a way that adding an MS2-binding loopshould not interfere with any Cas9 residues. Additionally, the proximityof the tetraloop and stem loop 2 sites to the DNA indicates thatlocalization to these locations could result in a high degree ofinteraction between the DNA and any recruited protein, such as atranscriptional activator. Thus, in some sgRNAs, nucleotidescorresponding to +13 to +16 and/or nucleotides corresponding to +53 to+56 of the guide RNA scaffold set forth in SEQ ID NO: 17 or SEQ ID NO:19 (or SEQ ID NO: 30 or 31) or corresponding residues when optimallyaligned with any of these scaffold/backbones are replaced by thedistinct RNA sequences capable of binding to one or more adaptorproteins or domains. Alternatively or additionally, adaptor-bindingsequences can be added to the 5′ end or the 3′ end of a guide RNA. Anexemplary guide RNA scaffold comprising MS2-binding loops in thetetraloop and stem loop 2 regions can comprise, consist essentially of,or consist of the sequence set forth in SEQ ID NO: 34. An exemplarygeneric single guide RNA comprising MS2-binding loops in the tetraloopand stem loop 2 regions can comprise, consist essentially of, or consistof the sequence set forth in SEQ ID NO: 35.

Guide RNAs can be provided in any form. For example, the gRNA can beprovided in the form of RNA, either as two molecules (separate crRNA andtracrRNA) or as one molecule (sgRNA), and optionally in the form of acomplex with a Cas protein. The gRNA can also be provided in the form ofDNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNAmolecule (sgRNA) or separate RNA molecules (e.g., separate crRNA andtracrRNA). In the latter case, the DNA encoding the gRNA can be providedas one DNA molecule or as separate DNA molecules encoding the crRNA andtracrRNA, respectively.

When a gRNA is provided in the form of DNA, the gRNA can be transiently,conditionally, or constitutively expressed in the cell. DNAs encodinggRNAs can be stably integrated into the genome of the cell and operablylinked to a promoter active in the cell. Alternatively, DNAs encodinggRNAs can be operably linked to a promoter in an expression construct.For example, the DNA encoding the gRNA can be in a vector comprising aheterologous nucleic acid, such as a nucleic acid encoding a Casprotein. Alternatively, it can be in a vector or a plasmid that isseparate from the vector comprising the nucleic acid encoding the Casprotein. Promoters that can be used in such expression constructsinclude promoters active, for example, in one or more of a eukaryoticcell, a human cell, a non-human cell, a mammalian cell, a non-humanmammalian cell, a rodent cell, a mouse cell, a rat cell, a pluripotentcell, an embryonic stem (ES) cell, an adult stem cell, a developmentallyrestricted progenitor cell, an induced pluripotent stem (iPS) cell, or aone-cell stage embryo. Such promoters can be, for example, conditionalpromoters, inducible promoters, constitutive promoters, ortissue-specific promoters. Such promoters can also be, for example,bidirectional promoters. Specific examples of suitable promoters includean RNA polymerase III promoter, such as a human U6 promoter, a rat U6polymerase III promoter, or a mouse U6 polymerase III promoter.

Alternatively, gRNAs can be prepared by various other methods. Forexample, gRNAs can be prepared by in vitro transcription using, forexample, T7 RNA polymerase (see, e.g., WO 2014/089290 and WO2014/065596, each of which is herein incorporated by reference in itsentirety for all purposes). Guide RNAs can also be a syntheticallyproduced molecule prepared by chemical synthesis. For example, a guideRNA can be chemically synthesized to include 2′-O-methyl analogs and 3′phosphorothioate internucleotide linkages at the first three 5′ and 3′terminal RNA residues.

Guide RNAs (or nucleic acids encoding guide RNAs) can be in compositionscomprising one or more guide RNAs (e.g., 1, 2, 3, 4, or more guide RNAs)and a carrier increasing the stability of the guide RNA (e.g.,prolonging the period under given conditions of storage (e.g., −20° C.,4° C., or ambient temperature) for which degradation products remainbelow a threshold, such below 0.5% by weight of the starting nucleicacid or protein; or increasing the stability in vivo). Non-limitingexamples of such carriers include poly(lactic acid) (PLA) microspheres,poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes,micelles, inverse micelles, lipid cochleates, and lipid microtubules.Such compositions can further comprise a Cas protein, such as a Cas9protein, or a nucleic acid encoding a Cas protein.

B. Guide RNA Target Sequences

Target DNAs for guide RNAs include nucleic acid sequences present in aDNA to which a DNA-targeting segment of a gRNA will bind, providedsufficient conditions for binding exist. Suitable DNA/RNA bindingconditions include physiological conditions normally present in a cell.Other suitable DNA/RNA binding conditions (e.g., conditions in acell-free system) are known in the art (see, e.g., Molecular Cloning: ALaboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press2001), herein incorporated by reference in its entirety for allpurposes). The strand of the target DNA that is complementary to andhybridizes with the gRNA can be called the “complementary strand,” andthe strand of the target DNA that is complementary to the “complementarystrand” (and is therefore not complementary to the Cas protein or gRNA)can be called “noncomplementary strand” or “template strand.”

The target DNA includes both the sequence on the complementary strand towhich the guide RNA hybridizes and the corresponding sequence on thenon-complementary strand (e.g., adjacent to the protospacer adjacentmotif (PAM)). The term “guide RNA target sequence” as used herein refersspecifically to the sequence on the non-complementary strandcorresponding to (i.e., the reverse complement of) the sequence to whichthe guide RNA hybridizes on the complementary strand. That is, the guideRNA target sequence refers to the sequence on the non-complementarystrand adjacent to the PAM (e.g., upstream or 5′ of the PAM in the caseof Cas9). A guide RNA target sequence is equivalent to the DNA-targetingsegment of a guide RNA, but with thymines instead of uracils. As oneexample, a guide RNA target sequence for an SpCas9 enzyme can refer tothe sequence upstream of the 5′-NGG-3′ PAM on the non-complementarystrand. A guide RNA is designed to have complementarity to thecomplementary strand of a target DNA, where hybridization between theDNA-targeting segment of the guide RNA and the complementary strand ofthe target DNA promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided that there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. If a guide RNA is referred to herein as targeting aguide RNA target sequence, what is meant is that the guide RNAhybridizes to the complementary strand sequence of the target DNA thatis the reverse complement of the guide RNA target sequence on thenon-complementary strand.

A target DNA or guide RNA target sequence can comprise anypolynucleotide, and can be located, for example, in the nucleus orcytoplasm of a cell or within an organelle of a cell, such as amitochondrion or chloroplast. A target DNA or guide RNA target sequencecan be any nucleic acid sequence endogenous or exogenous to a cell. Theguide RNA target sequence can be a sequence coding a gene product (e.g.,a protein) or a non-coding sequence (e.g., a regulatory sequence) or caninclude both.

For CRISPRa and SAM systems, it can be preferable for the targetsequence to be adjacent to the transcription start site of a gene. Forexample, the target sequence can be within 1000, 900, 800, 700, 600,500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 1 base pair of thetranscription start site. Optionally, the target sequence is within theregion 200 base pairs upstream of the transcription start site and 1base pair downstream of the transcription start site (−200 to +1).

Site-specific binding and cleavage of a target DNA by a Cas protein canoccur at locations determined by both (i) base-pairing complementaritybetween the guide RNA and the complementary strand of the target DNA and(ii) a short motif, called the protospacer adjacent motif (PAM), in thenon-complementary strand of the target DNA. The PAM can flank the guideRNA target sequence. Optionally, the guide RNA target sequence can beflanked on the 3′ end by the PAM (e.g., for Cas9). Alternatively, theguide RNA target sequence can be flanked on the 5′ end by the PAM (e.g.,for Cpf1). For example, the cleavage site of Cas proteins can be about 1to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs)upstream or downstream of the PAM sequence (e.g., within the guide RNAtarget sequence). In the case of SpCas9, the PAM sequence (i.e., on thenon-complementary strand) can be 5′-N₁GG-3′, where N₁ is any DNAnucleotide, and where the PAM is immediately 3′ of the guide RNA targetsequence on the non-complementary strand of the target DNA. As such, thesequence corresponding to the PAM on the complementary strand (i.e., thereverse complement) would be 5′-CCN₂-3′, where N₂ is any DNA nucleotideand is immediately 5′ of the sequence to which the DNA-targeting segmentof the guide RNA hybridizes on the complementary strand of the targetDNA. In some such cases, N₁ and N₂ can be complementary and the N₁-N₂base pair can be any base pair (e.g., N₁=C and N₂=G; N₁=G and N₂=C; N₁=Aand N₂=T; or N₁=T, and N₂=A). In the case of Cas9 from S. aureus, thePAM can be NNGRRT or NNGRR, where N can A, G, C, or T, and R can be G orA. In the case of Cas9 from C. jejuni, the PAM can be, for example,NNNNACAC or NNNNRYAC, where N can be A, G, C, or T, and R can be G or A.In some cases (e.g., for FnCpf1), the PAM sequence can be upstream ofthe 5′ end and have the sequence 5′-TTN-3′.

An example of a guide RNA target sequence is a 20-nucleotide DNAsequence immediately preceding an NGG motif recognized by an SpCas9protein. For example, two examples of guide RNA target sequences plusPAMs are GN₁₉NGG (SEQ ID NO: 25) or N₂₀NGG (SEQ ID NO: 26). See, e.g.,WO 2014/165825, herein incorporated by reference in its entirety for allpurposes. The guanine at the 5′ end can facilitate transcription by RNApolymerase in cells. Other examples of guide RNA target sequences plusPAMs can include two guanine nucleotides at the 5′ end (e.g., GGN₂₀NGG;SEQ ID NO: 27) to facilitate efficient transcription by T7 polymerase invitro. See, e.g., WO 2014/065596, herein incorporated by reference inits entirety for all purposes. Other guide RNA target sequences plusPAMs can have between 4-22 nucleotides in length of SEQ ID NOS: 25-27,including the 5′ G or GG and the 3′ GG or NGG. Yet other guide RNAtarget sequences plus PAMs can have between 14 and 20 nucleotides inlength of SEQ ID NOS: 25-27.

Formation of a CRISPR complex hybridized to a target DNA can result incleavage of one or both strands of the target DNA within or near theregion corresponding to the guide RNA target sequence (i.e., the guideRNA target sequence on the non-complementary strand of the target DNAand the reverse complement on the complementary strand to which theguide RNA hybridizes). For example, the cleavage site can be within theguide RNA target sequence (e.g., at a defined location relative to thePAM sequence). The “cleavage site” includes the position of a target DNAat which a Cas protein produces a single-strand break or a double-strandbreak. The cleavage site can be on only one strand (e.g., when a nickaseis used) or on both strands of a double-stranded DNA. Cleavage sites canbe at the same position on both strands (producing blunt ends; e.g.Cas9)) or can be at different sites on each strand (producing staggeredends (i.e., overhangs); e.g., Cpf1). Staggered ends can be produced, forexample, by using two Cas proteins, each of which produces asingle-strand break at a different cleavage site on a different strand,thereby producing a double-strand break. For example, a first nickasecan create a single-strand break on the first strand of double-strandedDNA (dsDNA), and a second nickase can create a single-strand break onthe second strand of dsDNA such that overhanging sequences are created.In some cases, the guide RNA target sequence or cleavage site of thenickase on the first strand is separated from the guide RNA targetsequence or cleavage site of the nickase on the second strand by atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250,500, or 1,000 base pairs.

IV. Methods of Screening for Genetic Modifiers of Tau Seeding orAggregation

The Cas/tau biosensor cell lines disclosed herein can be used in methodsof screening for genetic modifiers of tau seeding or aggregation. Suchmethods can comprise providing a population of Cas/tau biosensor cellsas disclosed elsewhere herein, introducing a library comprising aplurality of unique guide RNAs, and assessing tau seeding or aggregationin the targeted cells.

As one example, a method can comprise providing a population of Cas/taubiosensor cells (e.g., a population of cells comprising a Cas protein, afirst tau repeat domain linked to a first reporter, and a second taurepeat domain linked to a second reporter), introducing into thepopulation of cells a library comprising a plurality of unique guideRNAs that target a plurality of genes, and culturing the population ofcells to allow genome editing and expansion. The plurality of uniqueguide RNAs form complexes with the Cas protein, and the Cas proteincleaves the plurality of genes resulting in knockout of gene function toproduce a genetically modified population of cells. The geneticallymodified population of cells can then be contacted with a tau seedingagent to produce a seeded population of cells. The seeded population ofcells can be cultured to allow tau aggregates to form, whereinaggregates of the first tau repeat domain and the second tau repeatdomain form in a subset of the seeded population of cells to produce anaggregation-positive population of cells. Finally, abundance of each ofthe plurality of unique guide RNAs can be determined in theaggregation-positive population of cells relative to the population ofcells being cultured after introduction of the guide RNA library.Enrichment of a guide RNA in the aggregation-positive population ofcells relative to the population of cells being cultured afterintroduction of the guide RNA library indicates that the gene targetedby the guide RNA is a genetic modifier of tau aggregation, whereindisruption of the gene targeted by the guide RNA enhances tauaggregation, or is a candidate genetic modifier of tau aggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to enhance tauaggregation.

Similarly, the SAM/tau biosensor cell lines disclosed herein can be usedin methods of screening for genetic modifiers of tau seeding oraggregation. Such methods can comprise providing a population of SAM/taubiosensor cells as disclosed elsewhere herein, introducing a librarycomprising a plurality of unique guide RNAs, and assessing tau seedingor aggregation in the targeted cells.

As one example, a method can comprise providing a population of SAM/taubiosensor cells (e.g., a population of cells comprising a chimeric Casprotein comprising a nuclease-inactive Cas protein fused to one or moretranscriptional activation domains, a chimeric adaptor proteincomprising an adaptor protein fused to one or more transcriptionalactivation domains, a first tau repeat domain linked to a firstreporter, and a second tau repeat domain linked to a second reporter),introducing into the population of cells a library comprising aplurality of unique guide RNAs that target a plurality of genes, andculturing the population of cells to allow transcriptional activationand expansion. The plurality of unique guide RNAs form complexes withthe chimeric Cas protein and the chimeric adaptor protein, and thecomplexes activate transcription of the plurality of genes resulting inincreased gene expression and a modified population of cells. Themodified population of cells can then be contacted with a tau seedingagent to produce a seeded population of cells. The seeded population ofcells can be cultured to allow tau aggregates to form, whereinaggregates of the first tau repeat domain and the second tau repeatdomain form in a subset of the seeded population of cells to produce anaggregation-positive population of cells. Finally, abundance of each ofthe plurality of unique guide RNAs can be determined in theaggregation-positive population of cells relative to the population ofcells being cultured after introduction of the guide RNA library.Enrichment of a guide RNA in the aggregation-positive population ofcells relative to the population of cells being cultured afterintroduction of the guide RNA library indicates that the gene targetedby the guide RNA is a genetic modifier of tau aggregation, whereintranscriptional activation of the gene targeted by the guide RNAenhances tau aggregation, or is a candidate genetic modifier of tauaggregation (e.g., for further testing via secondary screens), whereintranscriptional activation of the gene targeted by the guide RNA isexpected to enhance tau aggregation.

The Cas/tau biosensor cells used in the method can be any of the Cas/taubiosensor cells disclosed elsewhere herein. Likewise, the SAM/taubiosensor cells used in the method can be any of the SAM/tau biosensorcells disclosed elsewhere herein. The first tau repeat domain and thesecond tau repeat domain can be different or can be similar or the same.The tau repeat domain can be any of the tau repeat domains disclosedelsewhere herein. For example, the first tau repeat domain and/or thesecond tau repeat domain can be a wild type tau repeat domain or cancomprise a pro-aggregation mutation (e.g., a pathogenic, pro-aggregationmutation), such as a tau P301S mutation. The first tau repeat domainand/or the second tau repeat domain can comprise a tau four-repeatdomain. As one specific example, the first tau repeat domain and/or thesecond tau repeat domain can comprise, consist essentially of, orconsist of SEQ ID NO: 11 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In onespecific example, the nucleic acid encoding the tau repeat domain cancomprise, consist essentially of, or consist of SEQ ID NO: 12 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 12, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 11.

The first tau repeat domain can be linked to the first reporter and thesecond tau repeat domain can be linked to the second reporter by anymeans. For example, the reporter can be fused to the tau repeat domain(e.g., as part of a fusion protein). The reporter proteins can be anypair of reporter proteins that produce a detectable signal when thefirst tau repeat domain linked to the first reporter is aggregated withthe second tau repeat domain linked to the second reporter. As oneexample, the first and second reporters can be a split luciferaseprotein. As another example, the first and second reporter proteins canbe a fluorescence resonance energy transfer (FRET) pair. FRET is aphysical phenomenon in which a donor fluorophore in its excited statenon-radiatively transfers its excitation energy to a neighboringacceptor fluorophore, thereby causing the acceptor to emit itscharacteristic fluorescence. Examples of FRET pairs (donor and acceptorfluorophores) are well known. See, e.g., Bajar et al. (2016) Sensors(Basel) 16(9):1488, herein incorporated by reference in its entirety forall purposes. As one specific example of a FRET pair, the first reportercan be cyan fluorescent protein (CFP) and the second reporter can beyellow fluorescent protein (YFP). As a specific example, the CFP cancomprise, consist essentially of, or consist of SEQ ID NO: 13 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 13. As another specific example, the YFP cancomprise, consist essentially of, or consist of SEQ ID NO: 15 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 15.

For the Cas/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the Cas protein cancomprise, consist essentially of, or consist of SEQ ID NO: 21 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 21.

One or more or all of the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter can be stably expressed in the population of cells.For example, nucleic acids encoding one or more or all of the Casprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can begenomically integrated in the population of cells. In one specificexample, the nucleic acid encoding the Cas protein can comprise, consistessentially of, or consist of SEQ ID NO: 22 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 22, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 21.

For the SAM/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the chimeric Cas proteincan comprise the nuclease-inactive Cas protein fused to a VP64transcriptional activation domain. For example, the chimeric Cas proteincan comprise from N-terminus to C-terminus: the nuclease-inactive Casprotein; a nuclear localization signal; and the VP64 transcriptionalactivator domain. As one specific example, the adaptor protein can be anMS2 coat protein, and the one or more transcriptional activation domainsin the chimeric adaptor protein can comprise a p65 transcriptionalactivation domain and an HSF1 transcriptional activation domain. Forexample, the chimeric adaptor protein can comprise from N-terminus toC-terminus: the MS2 coat protein; a nuclear localization signal; the p65transcriptional activation domain; and the HSF1 transcriptionalactivation domain. In one specific example, the nucleic acid encodingthe chimeric Cas protein can comprise, consist essentially of, orconsist of SEQ ID NO: 38 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38,optionally wherein the nucleic acid encodes a protein comprising,consisting essentially of, or consisting of SEQ ID NO: 36. In onespecific example, the nucleic acid encoding the chimeric adaptor proteincan comprise, consist essentially of, or consist of SEQ ID NO: 39 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 39, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 37.

One or more or all of the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can be stablyexpressed in the population of cells. For example, nucleic acidsencoding one or more or all of the chimeric Cas protein, the chimericadaptor protein, the first tau repeat domain linked to the firstreporter, and the second tau repeat domain linked to the second reportercan be genomically integrated in the population of cells.

As disclosed elsewhere herein, the cells can be any type of cells. Forexample, the cells can be eukaryotic cells, mammalian cells, or humancells (e.g., HEK293T cells or neuronal cells).

The plurality of unique guide RNAs can be introduced into the populationof cells by any known means. In some methods, the guide RNAs areintroduced into the population of cells by viral transduction, such asretroviral, adenoviral, or lentiviral transduction. In a specificexample, the guide RNAs can be introduced by lentiviral transduction.Each of the plurality of unique guide RNAs can be in a separate viralvector. The population of cells can be infected at any multiplicity ofinfection. For example, the multiplicity of infection can be betweenabout 0.1 and about 1.0, between about 0.1 and about 0.9, between about0.1 and about 0.8, between about 0.1 and about 0.7, between about 0.1and about 0.6, between about 0.1 and about 0.5, between about 0.1 andabout 0.4, or between about 0.1 and about 0.3. Alternatively, themultiplicity of infection can be less than about 1.0, less than about0.9, less than about 0.8, less than about 0.7, less than about 0.6, lessthan about 0.5, less than about 0.4, less than about 0.3, or less thanabout 0.2. In a specific example, the multiplicity of infection can beless than about 0.3.

The guide RNAs can be introduced into the population of cells togetherwith a selection marker or reporter gene to select for cells that havethe guide RNAs, and the method can further comprise selecting cells thatcomprise the selection marker or reporter gene. Examples of selectionmarkers and reporter genes are provided elsewhere herein. As oneexample, the selection marker can be one that imparts resistance to adrug, such as neomycin phosphotransferase, hygromycin Bphosphotransferase, puromycin-N-acetyltransferase, and blasticidin Sdeaminase. Another exemplary selection marker is bleomycin resistanceprotein, encoded by the Sh ble gene (Streptoalloteichus hindustanusbleomycin gene), which confers zeocin (phleomycin D1) resistance. Forexample, cells can be selected with a drug (e.g., puromycin) so thatonly cells transduced with a guide RNA construct are preserved for beingused to carry out screening. For example, the drug can be puromycin orzeocin (phleomycin D1).

In some methods, the plurality of unique guide RNAs are introduced at aconcentration selected such that a majority of the cells receive onlyone of the unique guide RNAs. For example, if the guide RNAs are beingintroduced by viral transduction, the cells can be infected at a lowmultiplicity of infection to ensure that most cells receive only oneviral construct with high probability. As one specific example, themultiplicity of infection can be less than about 0.3.

The population of cells into which the plurality of unique guide RNAs isintroduced can be any suitable number of cells. For example, thepopulation of cells can comprise greater than about 50, greater thanabout 100, greater than about 200, greater than about 300, greater thanabout 400, greater than about 500, greater than about 600, greater thanabout 700, greater than about 800, greater than about 900, or greaterthan about 1000 cells per unique guide RNA. In a specific example, thepopulation of cells comprises greater than about 300 cells or greaterthan about 500 cells per unique guide RNA.

The plurality of unique guide RNAs can target any number of genes. Forexample, the plurality of unique guide RNAs can target about 50 or moregenes, about 100 or more genes, about 200 or more genes, about 300 ormore genes, about 400 or more genes, about 500 or more genes, about 1000or more genes, about 2000 or more genes, about 3000 or more genes, about4000 or more genes, about 5000 or more genes, about 10000 or more genes,or about 20000 or more genes. In some methods, the guide RNAs can beselected to target genes in a particular signaling pathway. In somemethods, the library of unique guide RNAs is a genome-wide library.

The plurality of unique guide RNAs can target any number of sequenceswithin each individual targeted gene. In some methods, a plurality oftarget sequences are targeted on average in each of the targetedplurality of genes. For example, about 2 to about 10, about 2 to about9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2to about 5, about 2 to about 4, or about 2 to about 3 unique targetsequences can be targeted on average in each of the targeted pluralityof genes. For example, at least about 2, at least about 3, at leastabout 4, at least about 5, or at least about 6 unique target sequencescan be targeted on average in each of the targeted plurality of genes.As a specific example, about 6 target sequences can be targeted onaverage in each of the targeted plurality of genes. As another specificexample, about 3 to about 6 or about 4 to about 6 target sequences aretargeted on average in each of the targeted plurality of genes.

The guide RNAs can target any desired location in the target genes. Insome CRISPRn methods using the Cas/tau biosensor cells, each guide RNAtargets a constitutive exon if possible. In some methods, each guide RNAtargets a 5′ constitutive exon if possible. In some methods, each guideRNA targets a first exon, a second exon, or a third exon (from the 5′end of the gene) if possible. In some CRISPRa methods using the SAM/taubiosensor cells, each guide RNA can target a guide RNA target sequencewithin 200 bp upstream of a transcription start site, if possible. Insome CRISPRa methods using the SAM/tau biosensor cells, wherein eachguide RNA can comprise one or more adaptor-binding elements to which thechimeric adaptor protein can specifically bind. In one example, eachguide RNA comprises two adaptor-binding elements to which the chimericadaptor protein can specifically bind, optionally wherein a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. For example, theadaptor-binding element can comprise the sequence set forth in SEQ IDNO: 33. In a specific example, each of one or more guide RNAs is asingle guide RNA comprising a CRISPR RNA (crRNA) portion fused to atransactivating CRISPR RNA (tracrRNA) portion, and the first loop is thetetraloop corresponding to residues 13-16 of SEQ ID NO: 17, 19, 30, or31, and the second loop is the stem loop 2 corresponding to residues53-56 of SEQ ID NO: 17, 19, 30, or 31.

The step of culturing the population of cells to allow genome editingand expansion can be any suitable period of time. For example, theculturing can be for between about 2 days and about 10 days, betweenabout 3 days and about 9 days, between about 4 days and about 8 days,between about 5 days and about 7 days, or about 6 days. Likewise, thestep of culturing the population of cells to allow transcriptionalactivation and expansion can be any suitable period of time. Forexample, the culturing can be for between about 2 days and about 10days, between about 3 days and about 9 days, between about 4 days andabout 8 days, between about 5 days and about 7 days, or about 6 days.

Any suitable tau seeding agent can be used to produce a seededpopulation of cells. Suitable tau seeding agents are disclosed elsewhereherein. Some suitable seeding agents comprise a tau repeat domain thatcan be, for example, different from or similar to or the same as thefirst tau repeat domain and/or the second tau repeat domain. In oneexample, the seeding step comprises culturing the genetically modifiedpopulation of cells in the presence of conditioned medium harvested fromcultured tau-aggregation-positive cells in which a tau repeat domainstably presents in an aggregated state. For example, the conditionedmedium can have been harvested from confluent tau-aggregation-positivecells after being on the confluent cells for about 1 to about 7 days,about 2 to about 6 days, about 3 to about 5 days, or about 4 days. Theseeding step can comprise culturing the genetically modified populationof cells in any suitable ratio of conditioned medium to fresh medium.For example, it can comprise culturing the genetically modifiedpopulation of cells in about 90% conditioned medium and about 10% freshmedium, about 85% conditioned medium and about 15% fresh medium, about80% conditioned medium and about 20% fresh medium, about 75% conditionedmedium and about 25% fresh medium, about 70% conditioned medium andabout 30% fresh medium, about 65% conditioned medium and about 35% freshmedium, about 60% conditioned medium and about 40% fresh medium, about55% conditioned medium and about 45% fresh medium, about 50% conditionedmedium and about 50% fresh medium, about 45% conditioned medium andabout 55% fresh medium, about 40% conditioned medium and about 60% freshmedium, about 35% conditioned medium and about 65% fresh medium, about30% conditioned medium and about 70% fresh medium, about 25% conditionedmedium and about 75% fresh medium, about 20% conditioned medium andabout 80% fresh medium, about 15% conditioned medium and about 85% freshmedium, or about 10% conditioned medium and about 90% fresh medium. Inone example, it can comprise culturing the genetically modifiedpopulation of cells in a medium that comprises at least about 50%conditioned medium and no more than about 50% fresh medium. In aspecific example, it can comprise culturing the genetically modifiedpopulation of cells in about 75% conditioned medium and about 25% freshmedium. Optionally, the genetically modified population of cells is notco-cultured with the tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state.

The step of culturing the seeded population of cells to allow tauaggregates to form, wherein aggregates of the first tau repeat domainand the second tau repeat domain form in a subset of the seededpopulation of cells to produce an aggregation-positive population ofcells, can be any suitable length of time. For example, the culturingcan be for between about 1 day and about 7 days, between about 2 daysand about 6 days, between about 3 days and about 5 days, or about 4days. Aggregation can be determined by any suitable means, depending onthe reporters used. For example, in methods in which the first reporterand the second reporter are a fluorescence resonance energy transfer(FRET) pair, the aggregation-positive population of cells can beidentified by flow cytometry.

Abundance of guide RNAs can be determined by any suitable means. In aspecific example, abundance is determined by next-generation sequencing.Next-generation sequencing refers to non-Sanger-based high-throughputDNA sequencing technologies. For example, determining abundance of aguide RNA can comprise measuring read counts of the guide RNA.

In some methods, a guide RNA is considered enriched if the abundance ofthe guide RNA relative to the total population of the plurality ofunique guide RNAs is at least about 1.5-fold higher in theaggregation-positive population of cells relative to the population ofcells being cultured after introduction of the guide RNA library.Different enrichment thresholds can also be used. For example, anenrichment threshold can be set higher to be more stringent (e.g., atleast about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold,at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold,or at least about 2.5-fold). Alternatively, an enrichment threshold canbe set lower to be less stringent (e.g., at least about 1.4-fold, atleast about 1.3-fold, or at least about 1.2-fold).

In one example, the step of determining abundance can comprisedetermining abundance of the plurality of unique guide RNAs in theaggregation-positive population relative to the population of cellscultured after introduction of the guide RNA library at a first timepoint during the culturing and/or a second time point during theculturing. For example, the first time point can be at a first passageof culturing the population of cells, and the second time point can bein the middle of culturing the population of cells to allow genomeediting and expansion. For example, the first time point can be after asufficient amount of time for the guide RNAs to form complexes with theCas protein, and for the Cas protein to cleave the plurality of genesresulting in knockout of gene function (CRISPRn) or to transcriptionallyactivate the plurality of genes (CRISPRa). However, the first time pointshould ideally be at the first cell passage to determine the gRNAlibrary representation soon after infection (i.e., before furtherexpansion and genome editing) and to determine if each gRNArepresentation evolves from the first time point to the second timepoints and to any additional time points to a final time point. Thisallows ruling out enriched gRNAs/targets due to cell growth advantagesduring the course of the screen by verifying gRNA abundance is unchangedbetween the first and second time points. As a specific example, thefirst time point can be after about 1 day, about 2 days, about 3 days,or about 4 days of culturing and expansion, and the second time pointcan be after about 3 days, about 4 days, about 5 days, or about 6 daysof culturing and expansion. For example, the first time point can beafter about 3 days of culturing and expansion, and the second time pointcan be after about 6 days of culturing and expansion. In some methods, agene can then be considered a genetic modifier of tau aggregation,wherein disruption (CRISPRn) or transcriptional activation (CRISPRa) ofthe gene enhances (or is expected to enhance) tau aggregation, if theabundance of a guide RNA targeting the gene relative to the totalpopulation of the plurality of unique guide RNAs is at least 1.5-foldhigher (or higher than a different selected enrichment threshold) in theaggregation-positive population of cells relative to the population ofcells cultured after introduction of the guide RNA library at both thefirst time point and the second time point. Alternatively oradditionally, a gene can be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene enhances (or is expected to enhance) tauaggregation, if the abundance of at least two unique guide RNAstargeting the gene relative to the total population of the plurality ofunique guide RNAs is at least 1.5-fold higher (or higher than adifferent selected enrichment threshold) in the aggregation-positivepopulation of cells relative to the population of cells cultured afterintroduction of the guide RNA library at either the first time point orthe second time point.

In some CRISPRn methods, the following steps are taken to identify agene as a genetic modifier of tau aggregation, wherein disruption(CRISPRn) or transcriptional activation (CRISPRa) of the gene enhances(or is expected to enhance) tau aggregation. Likewise, in some CRISPRamethods, the following steps are taken to identify a gene as a geneticmodifier of tau aggregation, wherein transcriptional activation of thegene enhances tau aggregation. The first step comprises identifyingwhich of the plurality of unique guide RNAs are present in theaggregation-positive population of cells. The second step comprisescalculating the random chance of the guide RNAs identified being presentusing the formula nCn′*(x−n′)C(m−n)/xCm, where x is the variety ofunique guide RNAs introduced into the population of cells, m is thevariety of unique guide RNAs identified in step (1), n is the variety ofunique guide RNAs introduced into the population of cells that targetthe gene, and n′ is the variety of unique guide RNAs identified in step(1) that target the gene. The third step comprises calculating averageenrichment scores for the guide RNAs identified in step (1). Theenrichment score for a guide RNA is the relative abundance of the guideRNA in the aggregation-positive population of cells divided by therelative abundance of the guide RNA in the population of cells culturedafter introduction of the guide RNA library. The relative abundance isthe read count of the guide RNA divided by the read count of the totalpopulation of the plurality of unique guide RNAs. The fourth stepcomprises selecting the gene if a guide RNA targeting the gene issignificantly below the random chance of being present and above athreshold enrichment score. Possible threshold enrichment scores arediscussed above. As a specific example, the threshold enrichment scorecan be set at about 1.5-fold.

Variety when used in the phrase variety of unique guide RNAs means thenumber of unique guide RNA sequences. It is not the abundance, butrather the qualitative “present” or “not present.” Variety of uniqueguide RNAs means the number of unique guide RNA sequence. The variety ofunique guide RNA is determined by next generation sequencing (NGS) toidentify all the unique guide RNAs present in a cell population. It isdone by using two primers that recognize the constant regions of theviral vector to amplify the gRNA that is between the constant regionsand a primer that recognizes one constant region for sequencing. Eachunique guide RNA present in the sample will generate read counts usingthe sequencing primer. The NGS results will include the sequence andalso the number of reads corresponding to the sequence. The number ofreads will be used for the enrichment score calculation for each guideRNA, and the presence of each unique sequence will tell us which guideRNAs are present. For instance, if there are three unique guide RNAs fora gene before selection, and all three are retained post-selection, thenboth n and n′ are 3. These numbers are used for calculating thestatistics but not the actual read counts. However, the read counts foreach guide RNA (in one example, 100, 200, 50, which correspond to eachof the 3 unique guide RNAs) will be used for the calculation ofenrichment score.

V. Methods of Screening for Genetic Modifiers of Tau Aggregation thatPrevent Tau Aggregation

The Cas/tau biosensor cell lines disclosed herein can be used in methodsof screening for genetic modifiers of tau aggregation (e.g., thatprevent tau aggregation or are expected to prevent tau aggregation).Such methods can comprise providing a population of Cas/tau biosensorcells as disclosed elsewhere herein, introducing a library comprising aplurality of unique guide RNAs, and assessing tau aggregation in thetargeted cells.

As one example, a method can comprise providing a population of Cas/taubiosensor cells (e.g., a population of cells comprising a Cas protein, afirst tau repeat domain linked to a first reporter, and a second taurepeat domain linked to a second reporter), introducing into thepopulation of cells a library comprising a plurality of unique guideRNAs that target a plurality of genes, and culturing the population ofcells to allow genome editing and expansion. The plurality of uniqueguide RNAs form complexes with the Cas protein, and the Cas proteincleaves the plurality of genes resulting in knockout of gene function toproduce a genetically modified population of cells. The geneticallymodified population of cells can then be contacted with a tau seedingagent to produce a seeded population of cells. For example, the tauseeding agent can be a “maximum seeding” agent as described elsewhereherein, such as cell lysates from tau-aggregation-positive cells. Theseeded population of cells can be cultured to allow tau aggregates toform, wherein aggregates of the first tau repeat domain and the secondtau repeat domain form in a subset of the seeded population of cells toproduce an aggregation-positive population of cells and whereinaggregates do not form in a second subset of the seeded population ofcells to produce an aggregation-negative population of cells. Finally,abundance of each of the plurality of unique guide RNAs can bedetermined in the aggregation-positive population of cells relative tothe aggregation-negative population of cells and/or relative to thepopulation of cells after being seeded and/or relative to the populationof cells being cultured after introduction of the guide RNA library.Enrichment of a guide RNA in the aggregation-negative population ofcells relative to the aggregation-positive population of cells and/orrelative to the population of cells after being seeded and/or relativeto the population of cells being cultured after introduction of theguide RNA library indicates that the gene targeted by the guide RNA is agenetic modifier of tau aggregation, wherein disruption of the genetargeted by the guide RNA prevents tau aggregation, or is a candidategenetic modifier of tau aggregation (e.g., for further testing viasecondary screens), wherein disruption of the gene targeted by the guideRNA is expected to prevent tau aggregation. Likewise, depletion of aguide RNA in the aggregation-positive population of cells relative tothe aggregation-negative population of cells and/or relative to thepopulation of cells after being seeded and/or relative to the populationof cells being cultured after introduction of the guide RNA libraryindicates that the gene targeted by the guide RNA is a genetic modifierof tau aggregation, wherein disruption of the gene targeted by the guideRNA prevents tau aggregation, or is a candidate genetic modifier of tauaggregation (e.g., for further testing via secondary screens), whereindisruption of the gene targeted by the guide RNA is expected to preventtau aggregation. Enrichment of a guide RNA in the aggregation-positivepopulation of cells relative to the aggregation-negative population ofcells and/or relative to the population of cells after being seededand/or relative to the population of cells being cultured afterintroduction of the guide RNA library indicates that the gene targetedby the guide RNA is a genetic modifier of tau aggregation, whereindisruption of the gene targeted by the guide RNA promotes or enhancestau aggregation, or is a candidate genetic modifier of tau aggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to promote or enhance tauaggregation. Likewise, depletion of a guide RNA in theaggregation-negative population of cells relative to theaggregation-positive population of cells and/or relative to thepopulation of cells after being seeded and/or relative to the populationof cells being cultured after introduction of the guide RNA libraryindicates that the gene targeted by the guide RNA is a genetic modifierof tau aggregation, wherein disruption of the gene targeted by the guideRNA promotes or enhances tau aggregation, or is a candidate geneticmodifier of tau aggregation (e.g., for further testing via secondaryscreens), wherein disruption of the gene targeted by the guide RNA isexpected to promote or enhance tau aggregation.

Similarly, the SAM/tau biosensor cell lines disclosed herein can be usedin methods of screening for genetic modifiers of tau aggregation (e.g.,that prevent tau aggregation or are expected to prevent tauaggregation). Such methods can comprise providing a population ofSAM/tau biosensor cells as disclosed elsewhere herein, introducing alibrary comprising a plurality of unique guide RNAs, and assessing tauaggregation in the targeted cells.

As one example, a method can comprise providing a population of SAM/taubiosensor cells (e.g., a population of cells comprising a chimeric Casprotein comprising a nuclease-inactive Cas protein fused to one or moretranscriptional activation domains, a chimeric adaptor proteincomprising an adaptor protein fused to one or more transcriptionalactivation domains, a first tau repeat domain linked to a firstreporter, and a second tau repeat domain linked to a second reporter),introducing into the population of cells a library comprising aplurality of unique guide RNAs that target a plurality of genes, andculturing the population of cells to allow transcriptional activationand expansion. The plurality of unique guide RNAs form complexes withthe chimeric Cas protein and the chimeric adaptor protein, and thecomplexes activate transcription of the plurality of genes resulting inincreased gene expression and a modified population of cells. Themodified population of cells can then be contacted with a tau seedingagent to produce a seeded population of cells. For example, the tauseeding agent can be a “maximum seeding” agent as described elsewhereherein, such as cell lysates from tau-aggregation-positive cells. Theseeded population of cells can be cultured to allow tau aggregates toform, wherein aggregates of the first tau repeat domain and the secondtau repeat domain form in a subset of the seeded population of cells toproduce an aggregation-positive population of cells and whereinaggregates do not form in a second subset of the seeded population ofcells to produce an aggregation-negative population of cells. Finally,abundance of each of the plurality of unique guide RNAs can bedetermined in the aggregation-positive population of cells relative tothe aggregation-negative population of cells and/or relative to thepopulation of cells after being seeded and/or relative to the populationof cells being cultured after introduction of the guide RNA library.Enrichment of a guide RNA in the aggregation-negative population ofcells relative to the aggregation-positive population of cells and/orrelative to the population of cells after being seeded and/or relativeto the population of cells being cultured after introduction of theguide RNA library indicates that the gene targeted by the guide RNA is agenetic modifier of tau aggregation, wherein transcriptional activationof the gene targeted by the guide RNA prevents tau aggregation, or is acandidate genetic modifier of tau aggregation (e.g., for further testingvia secondary screens), wherein transcriptional activation of the genetargeted by the guide RNA is expected to prevent tau aggregation.Likewise, depletion of a guide RNA in the aggregation-positivepopulation of cells relative to the aggregation-negative population ofcells and/or relative to the population of cells after being seededand/or relative to the population of cells being cultured afterintroduction of the guide RNA library indicates that the gene targetedby the guide RNA is a genetic modifier of tau aggregation, whereintranscriptional activation of the gene targeted by the guide RNAprevents tau aggregation, or is a candidate genetic modifier of tauaggregation (e.g., for further testing via secondary screens), whereintranscriptional activation of the gene targeted by the guide RNA isexpected to prevent tau aggregation. Enrichment of a guide RNA in theaggregation-positive population of cells relative to theaggregation-negative population of cells and/or relative to thepopulation of cells after being seeded and/or relative to the populationof cells being cultured after introduction of the guide RNA libraryindicates that the gene targeted by the guide RNA is a genetic modifierof tau aggregation, wherein transcriptional activation of the genetargeted by the guide RNA promotes or enhances tau aggregation, or is acandidate genetic modifier of tau aggregation (e.g., for further testingvia secondary screens), wherein transcriptional activation of the genetargeted by the guide RNA is expected to promote or enhance tauaggregation. Likewise, depletion of a guide RNA in theaggregation-negative population of cells relative to theaggregation-positive population of cells and/or relative to thepopulation of cells after being seeded and/or relative to the populationof cells being cultured after introduction of the guide RNA libraryindicates that the gene targeted by the guide RNA is a genetic modifierof tau aggregation, wherein transcriptional activation of the genetargeted by the guide RNA promotes or enhances tau aggregation, or is acandidate genetic modifier of tau aggregation (e.g., for further testingvia secondary screens), wherein transcriptional activation of the genetargeted by the guide RNA is expected to promote or enhance tauaggregation.

The Cas/tau biosensor cells used in the method can be any of the Cas/taubiosensor cells disclosed elsewhere herein. Likewise, the SAM/taubiosensor cells used in the method can be any of the SAM/tau biosensorcells disclosed elsewhere herein. The first tau repeat domain and thesecond tau repeat domain can be different or can be similar or the same.The tau repeat domain can be any of the tau repeat domains disclosedelsewhere herein. For example, the first tau repeat domain and/or thesecond tau repeat domain can be a wild type tau repeat domain or cancomprise a pro-aggregation mutation (e.g., a pathogenic, pro-aggregationmutation), such as a tau P301S mutation. The first tau repeat domainand/or the second tau repeat domain can comprise a tau four-repeatdomain. As one specific example, the first tau repeat domain and/or thesecond tau repeat domain can comprise, consist essentially of, orconsist of SEQ ID NO: 11 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In onespecific example, the nucleic acid encoding the tau repeat domain cancomprise, consist essentially of, or consist of SEQ ID NO: 12 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 12, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 11.

The first tau repeat domain can be linked to the first reporter and thesecond tau repeat domain can be linked to the second reporter by anymeans. For example, the reporter can be fused to the tau repeat domain(e.g., as part of a fusion protein). The reporter proteins can be anypair of reporter proteins that produce a detectable signal when thefirst tau repeat domain linked to the first reporter is aggregated withthe second tau repeat domain linked to the second reporter. As oneexample, the first and second reporters can be a split luciferaseprotein. As another example, the first and second reporter proteins canbe a fluorescence resonance energy transfer (FRET) pair. FRET is aphysical phenomenon in which a donor fluorophore in its excited statenon-radiatively transfers its excitation energy to a neighboringacceptor fluorophore, thereby causing the acceptor to emit itscharacteristic fluorescence. Examples of FRET pairs (donor and acceptorfluorophores) are well known. See, e.g., Bajar et al. (2016) Sensors(Basel) 16(9):1488, herein incorporated by reference in its entirety forall purposes. As one specific example of a FRET pair, the first reportercan be cyan fluorescent protein (CFP) and the second reporter can beyellow fluorescent protein (YFP). As a specific example, the CFP cancomprise, consist essentially of, or consist of SEQ ID NO: 13 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 13. As another specific example, the YFP cancomprise, consist essentially of, or consist of SEQ ID NO: 15 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 15.

For the Cas/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the Cas protein cancomprise, consist essentially of, or consist of SEQ ID NO: 21 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 21.

One or more or all of the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter can be stably expressed in the population of cells.For example, nucleic acids encoding one or more or all of the Casprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can begenomically integrated in the population of cells. In one specificexample, the nucleic acid encoding the Cas protein can comprise, consistessentially of, or consist of SEQ ID NO: 22 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 22, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 21.

For the SAM/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the chimeric Cas proteincan comprise the nuclease-inactive Cas protein fused to a VP64transcriptional activation domain. For example, the chimeric Cas proteincan comprise from N-terminus to C-terminus: the nuclease-inactive Casprotein; a nuclear localization signal; and the VP64 transcriptionalactivator domain. As one specific example, the adaptor protein can be anMS2 coat protein, and the one or more transcriptional activation domainsin the chimeric adaptor protein can comprise a p65 transcriptionalactivation domain and an HSF1 transcriptional activation domain. Forexample, the chimeric adaptor protein can comprise from N-terminus toC-terminus: the MS2 coat protein; a nuclear localization signal; the p65transcriptional activation domain; and the HSF1 transcriptionalactivation domain. In one specific example, the nucleic acid encodingthe chimeric Cas protein can comprise, consist essentially of, orconsist of SEQ ID NO: 38 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38,optionally wherein the nucleic acid encodes a protein comprising,consisting essentially of, or consisting of SEQ ID NO: 36. In onespecific example, the nucleic acid encoding the chimeric adaptor proteincan comprise, consist essentially of, or consist of SEQ ID NO: 39 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 39, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 37.

One or more or all of the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can be stablyexpressed in the population of cells. For example, nucleic acidsencoding one or more or all of the chimeric Cas protein, the chimericadaptor protein, the first tau repeat domain linked to the firstreporter, and the second tau repeat domain linked to the second reportercan be genomically integrated in the population of cells.

As disclosed elsewhere herein, the cells can be any type of cells. Forexample, the cells can be eukaryotic cells, mammalian cells, or humancells (e.g., HEK293T cells or neuronal cells).

The plurality of unique guide RNAs can be introduced into the populationof cells by any known means. In some methods, the guide RNAs areintroduced into the population of cells by viral transduction, such asretroviral, adenoviral, or lentiviral transduction. In a specificexample, the guide RNAs can be introduced by lentiviral transduction.Each of the plurality of unique guide RNAs can be in a separate viralvector. The population of cells can be infected at any multiplicity ofinfection. For example, the multiplicity of infection can be betweenabout 0.1 and about 1.0, between about 0.1 and about 0.9, between about0.1 and about 0.8, between about 0.1 and about 0.7, between about 0.1and about 0.6, between about 0.1 and about 0.5, between about 0.1 andabout 0.4, or between about 0.1 and about 0.3. Alternatively, themultiplicity of infection can be less than about 1.0, less than about0.9, less than about 0.8, less than about 0.7, less than about 0.6, lessthan about 0.5, less than about 0.4, less than about 0.3, or less thanabout 0.2. In a specific example, the multiplicity of infection can beless than about 0.3.

The guide RNAs can be introduced into the population of cells togetherwith a selection marker or reporter gene to select for cells that havethe guide RNAs, and the method can further comprise selecting cells thatcomprise the selection marker or reporter gene. Examples of selectionmarkers and reporter genes are provided elsewhere herein. As oneexample, the selection marker can be one that imparts resistance to adrug, such as neomycin phosphotransferase, hygromycin Bphosphotransferase, puromycin-N-acetyltransferase, and blasticidin Sdeaminase. Another exemplary selection marker is bleomycin resistanceprotein, encoded by the Sh ble gene (Streptoalloteichus hindustanusbleomycin gene), which confers zeocin (phleomycin D1) resistance. Forexample, cells can be selected with a drug (e.g., puromycin) so thatonly cells transduced with a guide RNA construct are preserved for beingused to carry out screening. For example, the drug can be puromycin orzeocin (phleomycin D1).

In some methods, the plurality of unique guide RNAs are introduced at aconcentration selected such that a majority of the cells receive onlyone of the unique guide RNAs. For example, if the guide RNAs are beingintroduced by viral transduction, the cells can be infected at a lowmultiplicity of infection to ensure that most cells receive only oneviral construct with high probability. As one specific example, themultiplicity of infection can be less than about 0.3.

The population of cells into which the plurality of unique guide RNAs isintroduced can be any suitable number of cells. For example, thepopulation of cells can comprise greater than about 50, greater thanabout 100, greater than about 200, greater than about 300, greater thanabout 400, greater than about 500, greater than about 600, greater thanabout 700, greater than about 800, greater than about 900, or greaterthan about 1000 cells per unique guide RNA. In a specific example, thepopulation of cells comprises greater than about 300 cells or greaterthan about 500 cells per unique guide RNA.

The plurality of unique guide RNAs can target any number of genes. Forexample, the plurality of unique guide RNAs can target about 50 or moregenes, about 100 or more genes, about 200 or more genes, about 300 ormore genes, about 400 or more genes, about 500 or more genes, about 1000or more genes, about 2000 or more genes, about 3000 or more genes, about4000 or more genes, about 5000 or more genes, about 10000 or more genes,or about 20000 or more genes. In some methods, the guide RNAs can beselected to target genes in a particular signaling pathway. In somemethods, the library of unique guide RNAs is a genome-wide library.

The plurality of unique guide RNAs can target any number of sequenceswithin each individual targeted gene. In some methods, a plurality oftarget sequences are targeted on average in each of the targetedplurality of genes. For example, about 2 to about 10, about 2 to about9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2to about 5, about 2 to about 4, or about 2 to about 3 unique targetsequences can be targeted on average in each of the targeted pluralityof genes. For example, at least about 2, at least about 3, at leastabout 4, at least about 5, or at least about 6 unique target sequencescan be targeted on average in each of the targeted plurality of genes.As a specific example, about 6 target sequences can be targeted onaverage in each of the targeted plurality of genes. As another specificexample, about 3 to about 6 or about 4 to about 6 target sequences aretargeted on average in each of the targeted plurality of genes.

The guide RNAs can target any desired location in the target genes. Insome CRISPRn methods using the Cas/tau biosensor cells, each guide RNAtargets a constitutive exon if possible. In some methods, each guide RNAtargets a 5′ constitutive exon if possible. In some methods, each guideRNA targets a first exon, a second exon, or a third exon (from the 5′end of the gene) if possible. In some CRISPRa methods using the SAM/taubiosensor cells, each guide RNA can target a guide RNA target sequencewithin 200 bp upstream of a transcription start site, if possible. Insome CRISPRa methods using the SAM/tau biosensor cells, wherein eachguide RNA can comprise one or more adaptor-binding elements to which thechimeric adaptor protein can specifically bind. In one example, eachguide RNA comprises two adaptor-binding elements to which the chimericadaptor protein can specifically bind, optionally wherein a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. For example, theadaptor-binding element can comprise the sequence set forth in SEQ IDNO: 33. In a specific example, each of one or more guide RNAs is asingle guide RNA comprising a CRISPR RNA (crRNA) portion fused to atransactivating CRISPR RNA (tracrRNA) portion, and the first loop is thetetraloop corresponding to residues 13-16 of SEQ ID NO: 17, 19, 30, or31, and the second loop is the stem loop 2 corresponding to residues53-56 of SEQ ID NO: 17, 19, 30, or 31.

The step of culturing the population of cells to allow genome editingand expansion can be any suitable period of time. For example, theculturing can be for between about 2 days and about 15 days, betweenabout 3 days and about 13 days, between about 5 days and about 12 days,between about 7 days and about 11 days, or about 7 days or about 11days. As another example, the culturing can be for between about 2 daysand about 14 days, between about 3 days and about 12 days, between about5 days and about 11 days, between about 7 days and about 10 days, orabout 7 days or about 10 days. Likewise, the step of culturing thepopulation of cells to allow transcriptional activation and expansioncan be any suitable period of time. For example, the culturing can befor between about 2 days and about 15 days, between about 3 days andabout 13 days, between about 5 days and about 12 days, between about 7days and about 11 days, or about 7 days or about 11 days. Likewise, thestep of culturing the population of cells to allow transcriptionalactivation and expansion can be any suitable period of time. Forexample, the culturing can be for between about 2 days and about 14days, between about 3 days and about 12 days, between about 5 days andabout 11 days, between about 7 days and about 10 days, or about 7 daysor about 10 days.

Any suitable tau seeding agent can be used to produce a seededpopulation of cells. Suitable tau seeding agents are disclosed elsewhereherein. Some suitable seeding agents comprise a tau repeat domain thatcan be, for example, different from or similar to or the same as thefirst tau repeat domain and/or the second tau repeat domain. In oneexample, the seeding step comprises culturing the genetically modifiedpopulation of cells in the presence of comprising a cell lysate fromcultured tau-aggregation-positive cells in which a tau repeat domainstably presents in an aggregated state. Optionally, the geneticallymodified population of cells is not co-cultured with thetau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state.

The amount or concentration of the cell lysate in the medium can be anysuitable amount or concentration. For example, the concentration of celllysate in the medium can be between about 0.1 μg/mL and about 50 μg/mL,between about 0.1 μg/mL and about 25 μg/mL, between about 0.1 μg/mL andabout 10 μg/mL, between about 0.1 μg/mL and about 5 μg/mL, between about0.1 μg/mL and about 4.5 μg/mL, between about 0.1 μg/mL and about 4μg/mL, between about 0.1 μg/mL and about 3.5 μg/mL, between about 0.1μg/mL and about 3 μg/mL, between about 0.1 μg/mL and about 2.5 μg/mL,between about 0.1 μg/mL and about 2 μg/mL, between about 0.1 μg/mL andabout 1.5 μg/mL, between about 0.1 μg/mL and about 1 μg/mL, betweenabout 0.5 μg/mL and about 50 μg/mL, between about 0.5 μg/mL and about 25μg/mL, between about 0.5 μg/mL and about 10 μg/mL, between about 0.5μg/mL and about 5 μg/mL, between about 0.5 μg/mL and about 4.5 μg/mL,between about 0.5 μg/mL and about 4 μg/mL, between about 0.5 μg/mL andabout 3.5 μg/mL, between about 0.5 μg/mL and about 3 μg/mL, betweenabout 0.5 μg/mL and about 2.5 μg/mL, between about 0.5 μg/mL and about 2μg/mL, between about 0.5 μg/mL and about 1.5 μg/mL, between about 0.5μg/mL and about 1 μg/mL, between about 1 μg/mL and about 50 μg/mL,between about 1 μg/mL and about 25 μg/mL, between about 1 μg/mL andabout 10 μg/mL, between about 1 μg/mL and about 5 μg/mL, between about 1μg/mL and about 4.5 μg/mL, between about 1 μg/mL and about 4 μg/mL,between about 1 μg/mL and about 3.5 μg/mL, between about 1 μg/mL andabout 3 μg/mL, between about 1 μg/mL and about 2.5 μg/mL, between about1 μg/mL and about 2 μg/mL, between about 1 μg/mL and about 1.5 μg/mL,between about 1.5 μg/mL and about 50 μg/mL, between about 1.5 μg/mL andabout 25 μg/mL, between about 1.5 μg/mL and about 10 μg/mL, betweenabout 1.5 μg/mL and about 5 μg/mL, between about 1.5 μg/mL and about 4.5μg/mL, between about 1.5 μg/mL and about 4 μg/mL, between about 1.5μg/mL and about 3.5 μg/mL, between about 1.5 μg/mL and about 3 μg/mL,between about 1.5 μg/mL and about 2.5 μg/mL, between about 1.5 μg/mL andabout 2 μg/mL, between about 2 μg/mL and about 50 μg/mL, between about 2μg/mL and about 25 μg/mL, between about 2 μg/mL and about 10 μg/mL,between about 2 μg/mL and about 5 μg/mL, between about 2 μg/mL and about4.5 μg/mL, between about 2 μg/mL and about 4 μg/mL, between about 2μg/mL and about 3.5 μg/mL, between about 2 μg/mL and about 3 μg/mL,between about 2 μg/mL and about 2.5 μg/mL, between about 2.5 μg/mL andabout 50 μg/mL, between about 2.5 μg/mL and about 25 μg/mL, betweenabout 2.5 μg/mL and about 10 μg/mL, between about 2.5 μg/mL and about 5μg/mL, between about 2.5 μg/mL and about 4.5 μg/mL, between about 2.5μg/mL and about 4 μg/mL, between about 2.5 μg/mL and about 3.5 μg/mL, orbetween about 2.5 μg/mL and about 3 μg/mL of medium (e.g., fresh culturemedium). For example, the cell lysate in the culture medium can be at aconcentration of between about 1 μg/mL and about 5 μg/mL or can be at aconcentration of about 1.5 μg/mL, about 2 μg/mL, about 2.5 μg/mL, about3 μg/mL, about 3.5 μg/mL, about 4 g/mL, about 4.5 μg/mL, or about 5μg/mL. Optionally, the cell lysate can be in a buffer, such asphosphate-buffered saline. Optionally, the buffer can comprise proteaseinhibitors. Examples of protease inhibitors include, but are not limitedto, AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A, andethylenediaminetetracetic acid (EDTA). The buffer can comprise any ofthese inhibitors or any combination thereof (e.g., the buffer cancomprise all of these protease inhibitors).

The cells for producing the lysate can be collected in a buffer, such asphosphate-buffered saline. Optionally, the buffer can comprise proteaseinhibitors. Examples of protease inhibitors include, but are not limitedto, AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A, andethylenediaminetetracetic acid (EDTA). The buffer can comprise any ofthese inhibitors or any combination thereof (e.g., the buffer cancomprise all of these protease inhibitors).

The cell lysate can, for example, be collected by sonicating thetau-aggregation-positive cells (e.g., cells collected in a buffer andprotease inhibitors as described above) for any suitable amount of time.For example, the cells can be sonicated for between about 1 minute andabout 6 minutes, between about 1 minute and about 5 minutes, betweenabout 1 minute and about 4 minutes, between about 1 minute and about 3minutes, between about 2 minutes and about 6 minutes, between about 2minutes and about 5 minutes, between about 2 minutes and about 4minutes, between about 2 minutes and about 3 minutes, between about 2minutes and about 6 minutes, between about 3 minutes and about 5minutes, or between about 3 minutes and about 4 minutes. For example,the cells can be sonicated for between about 2 minutes and about 4minutes or for about 3 minutes.

Optionally, the medium comprises lipofectamine or liposomes (e.g.,cationic liposomes) or phospholipids or another transfection agent.Optionally, the medium comprises lipofectamine. Optionally, the mediumdoes not comprise lipofectamine or liposomes (e.g., cationic liposomes)or phospholipids or another transfection agent. Optionally, the mediumdoes not comprise lipofectamine. The amount or concentration of thelipofectamine or liposomes (e.g., cationic liposomes) or phospholipidsor other transfection agent in the medium can be any suitable amount orconcentration. For example, the concentration of lipofectamine orliposomes (e.g., cationic liposomes) or phospholipids or othertransfection agent in the medium can be between about 0.5 μL/mL to about10 μL/mL, between about 0.5 μL/mL to about 5 μL/mL, between about 0.5μL/mL to about 4.5 μL/mL, between about 0.5 μL/mL to about 4 μL/mL,between about 0.5 μL/mL to about 3.5 μL/mL, between about 0.5 μL/mL toabout 3 μL/mL, between about 0.5 μL/mL to about 2.5 μL/mL, between about0.5 μL/mL to about 2 μL/mL, between about 0.5 μL/mL to about 1.5 μL/mL,between about 0.5 μL/mL to about 1 μL/mL, between about 1 μL/mL to about10 μL/mL, between about 1 μL/mL to about 5 μL/mL, between about 1 μL/mLto about 4.5 μL/mL, between about 1 μL/mL to about 4 μL/mL, betweenabout 1 L/mL to about 3.5 μL/mL, between about 1 μL/mL to about 3 μL/mL,between about 1 μL/mL to about 2.5 μL/mL, between about 1 μL/mL to about2 μL/mL, between about 1 μL/mL to about 1.5 μL/mL, between about 1.5μL/mL to about 10 μL/mL, between about 1.5 μL/mL to about 5 L/mL,between about 1.5 μL/mL to about 4.5 μL/mL, between about 1.5 μL/mL toabout 4 L/mL, between about 1.5 μL/mL to about 3.5 μL/mL, between about1.5 μL/mL to about 3 L/mL, between about 1.5 μL/mL to about 2.5 μL/mL,between about 1.5 μL/mL to about 2 L/mL, between about 2 μL/mL to about10 μL/mL, between about 2 μL/mL to about 5 μL/mL, between about 2 μL/mLto about 4.5 μL/mL, between about 2 μL/mL to about 4 μL/mL, betweenabout 2 μL/mL to about 3.5 μL/mL, between about 2 μL/mL to about 3μL/mL, or between about 2 μL/mL to about 2.5 μL/mL of medium (e.g.,fresh medium). For example, the concentration of lipofectamine orliposomes (e.g., cationic liposomes) or phospholipids or othertransfection agent in the medium can be between about 1.5 μL/mL andabout 4 μL/mL or it can be about 1.5 L/mL, about 2 μL/mL, about 2.5μL/mL, about 3 μL/mL, about 3.5 μL/mL, or about 4 μL/mL.

The step of culturing the seeded population of cells to allow tauaggregates to form, wherein aggregates of the first tau repeat domainand the second tau repeat domain form in a subset of the seededpopulation of cells to produce an aggregation-positive population ofcells, can be any suitable length of time. For example, the culturingcan be for between about 1 day and about 7 days, between about 2 daysand about 6 days, between about 3 days and about 5 days, between about 1day and about 3 days, or about 2 days. Aggregation can be determined byany suitable means, depending on the reporters used. For example, inmethods in which the first reporter and the second reporter are afluorescence resonance energy transfer (FRET) pair, theaggregation-positive population of cells can be identified by flowcytometry.

Abundance of guide RNAs can be determined by any suitable means. In aspecific example, abundance is determined by next-generation sequencing.Next-generation sequencing refers to non-Sanger-based high-throughputDNA sequencing technologies. For example, determining abundance of aguide RNA can comprise measuring read counts of the guide RNA.

In some methods, a guide RNA is considered enriched inaggregation-negative cells if the abundance of the guide RNA relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-negative population of cells relativeto the aggregation-positive population of cells and/or the seededpopulation of cells. In some methods, a guide RNA is considered depletedin aggregation-positive cells if the abundance of the guide RNA relativeto the total population of the plurality of unique guide RNAs is atleast 1.5-fold lower in the aggregation-positive population of cellsrelative to the aggregation-negative population of cells and/or theseeded population of cells. Different enrichment/depletion thresholdscan also be used. For example, an enrichment/depletion threshold can beset higher to be more stringent (e.g., at least about 1.6-fold, at leastabout 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, atleast about 2-fold, at least about 2.5-fold, or at least about2.5-fold). Alternatively, an enrichment/depletion threshold can be setlower to be less stringent (e.g., at least about 1.4-fold, at leastabout 1.3-fold, or at least about 1.2-fold).

Alternatively, in some methods, a guide RNA is considered enriched inaggregation-positive cells if the abundance of the guide RNA relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-positive population of cells relativeto the aggregation-negative population of cells and/or the seededpopulation of cells. In some methods, a guide RNA is considered depletedin aggregation-negative cells if the abundance of the guide RNA relativeto the total population of the plurality of unique guide RNAs is atleast 1.5-fold lower in the aggregation-negative population of cellsrelative to the aggregation-positive population of cells and/or theseeded population of cells. Different enrichment/depletion thresholdscan also be used. For example, an enrichment/depletion threshold can beset higher to be more stringent (e.g., at least about 1.6-fold, at leastabout 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, atleast about 2-fold, at least about 2.5-fold, or at least about2.5-fold). Alternatively, an enrichment/depletion threshold can be setlower to be less stringent (e.g., at least about 1.4-fold, at leastabout 1.3-fold, or at least about 1.2-fold).

In one example, the step of determining abundance can comprisedetermining abundance of the plurality of unique guide RNAs in theaggregation-positive population relative to the aggregation-negativepopulation and/or relative to the population of cells cultured afterintroduction of the guide RNA library at a first time point and/orrelative to the seeded population of cells at a second time point.Likewise, the step of determining abundance can comprise determiningabundance of the plurality of unique guide RNAs in theaggregation-negative population relative to the aggregation-positivepopulation and/or relative to the population of cells cultured afterintroduction of the guide RNA library at a first time point and/orrelative to the seeded population of cells at a second time point. Forexample, the first time point can be at a first passage of culturing thepopulation of cells or in the middle of culturing the population ofcells to allow genome editing and expansion. For example, the first timepoint can be after a sufficient amount of time for the guide RNAs toform complexes with the Cas protein, and for the Cas protein to cleavethe plurality of genes resulting in knockout of gene function (CRISPRn)or to transcriptionally activate the plurality of genes (CRISPRa).However, in some cases, the first time point should ideally be at thefirst cell passage to determine the gRNA library representation soonafter infection (i.e., before further expansion and genome editing) andto determine if each gRNA representation evolves from the first timepoint to the second time points and to any additional time points to afinal time point. This allows ruling out enriched gRNAs/targets due tocell growth advantages during the course of the screen by verifying gRNAabundance is unchanged between the first and second time points. As aspecific example, the first time point can be after about 1 day, about 2days, about 3 days, or about 4 days of culturing and expansion (e.g., atabout 3 days of culture and expansion), and the second time point can beafter about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, or about 11 days of culturing and expansion. Forexample, the first time point can be after about 3 days of culturing andexpansion, and the second time point can be after about 7 days or about10 days of culturing and expansion.

In some methods, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene prevents (or is expected to prevent) tauaggregation, if the abundance of a guide RNA targeting the gene relativeto the total population of the plurality of unique guide RNAs is atleast 1.5-fold higher in the aggregation-negative population of cellsrelative to the aggregation-positive population of cells, the culturedpopulation of cells at the first time point, and the seeded populationof cells at the second time point. Alternatively or additionally, a genecan then be considered a genetic modifier of tau aggregation, whereindisruption (CRISPRn) or transcriptional activation (CRISPRa) of the geneprevents (or is expected to prevent) tau aggregation, if the abundanceof a guide RNA targeting the gene relative to the total population ofthe plurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-negative population of cells relative to theaggregation-positive population of cells and the seeded population ofcells at the second time point. Alternatively or additionally, a genecan then be considered a genetic modifier of tau aggregation, whereindisruption (CRISPRn) or transcriptional activation (CRISPRa) of the geneprevents (or is expected to prevent) tau aggregation, if the abundanceof a guide RNA targeting the gene relative to the total population ofthe plurality of unique guide RNAs is at least 1.5-fold lower in theaggregation-positive population of cells relative to theaggregation-negative population of cells, the cultured population ofcells at the first time point, and the seeded population of cells at thesecond time point. Alternatively or additionally, a gene can then beconsidered a genetic modifier of tau aggregation, wherein disruption(CRISPRn) or transcriptional activation (CRISPRa) of the gene prevents(or is expected to prevent) tau aggregation, if the abundance of a guideRNA targeting the gene relative to the total population of the pluralityof unique guide RNAs is at least 1.5-fold lower in theaggregation-positive population of cells relative to theaggregation-negative population of cells and the seeded population ofcells at the second time point.

Alternatively, in one example, the step of determining abundance cancomprise determining abundance of the plurality of unique guide RNAs inthe aggregation-negative population relative to the aggregation-positivepopulation and/or relative to the population of cells cultured afterintroduction of the guide RNA library at a first time point and/orrelative to the seeded population of cells at a second time point.Likewise, the step of determining abundance can comprise determiningabundance of the plurality of unique guide RNAs in theaggregation-positive population relative to the aggregation-negativepopulation and/or relative to the population of cells cultured afterintroduction of the guide RNA library at a first time point and/orrelative to the seeded population of cells at a second time point. Forexample, the first time point can be at a first passage of culturing thepopulation of cells or in the middle of culturing the population ofcells to allow genome editing and expansion. For example, the first timepoint can be after a sufficient amount of time for the guide RNAs toform complexes with the Cas protein, and for the Cas protein to cleavethe plurality of genes resulting in knockout of gene function (CRISPRn)or to transcriptionally activate the plurality of genes (CRISPRa).However, in some cases, the first time point should ideally be at thefirst cell passage to determine the gRNA library representation soonafter infection (i.e., before further expansion and genome editing) andto determine if each gRNA representation evolves from the first timepoint to the second time points and to any additional time points to afinal time point. This allows ruling out enriched gRNAs/targets due tocell growth advantages during the course of the screen by verifying gRNAabundance is unchanged between the first and second time points. As aspecific example, the first time point can be after about 1 day, about 2days, about 3 days, or about 4 days of culturing and expansion (e.g., atabout 3 days of culture and expansion), and the second time point can beafter about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, or about 11 days of culturing and expansion. Forexample, the first time point can be after about 3 days of culturing andexpansion, and the second time point can be after about 7 days or about10 days of culturing and expansion.

In some methods, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene promotes or enhances tau aggregation (or isexpected to promote or enhance tau aggregation), if the abundance of aguide RNA targeting the gene relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells relative to theaggregation-negative population of cells, the cultured population ofcells at the first time point, and the seeded population of cells at thesecond time point. Alternatively or additionally, a gene can then beconsidered a genetic modifier of tau aggregation, wherein disruption(CRISPRn) or transcriptional activation (CRISPRa) of the gene promotesor enhances tau aggregation (or is expected to promote or enhance tauaggregation), if the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold higher in the aggregation-positive population ofcells relative to the aggregation-negative population of cells and theseeded population of cells at the second time point. Alternatively oradditionally, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene promotes or enhances tau aggregation (or isexpected to promote or enhance tau aggregation), if the abundance of aguide RNA targeting the gene relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold lower in theaggregation-negative population of cells relative to theaggregation-positive population of cells, the cultured population ofcells at the first time point, and the seeded population of cells at thesecond time point. Alternatively or additionally, a gene can then beconsidered a genetic modifier of tau aggregation, wherein disruption(CRISPRn) or transcriptional activation (CRISPRa) of the gene promotesor enhances tau aggregation (or is expected to promote or enhance tauaggregation), if the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-negative population ofcells relative to the aggregation-positive population of cells and theseeded population of cells at the second time point.

In some CRISPRn methods, the following steps are taken to identify agene as a genetic modifier of tau aggregation, wherein disruption of thegene prevents (or is expected to prevent) tau aggregation. Likewise, insome CRISPRa methods, the following steps are taken to identify a geneas a genetic modifier of tau aggregation, wherein transcriptionalactivation of the gene prevents (or is expected to prevent) tauaggregation. The first step comprises identifying which of the pluralityof unique guide RNAs are present in the aggregation-negative populationof cells. The second step comprises calculating the random chance of theguide RNAs identified being present using the formulanCn′*(x−n′)C(m−n)/xCm, where x is the variety of unique guide RNAsintroduced into the population of cells, m is the variety of uniqueguide RNAs identified in step (1), n is the variety of unique guide RNAsintroduced into the population of cells that target the gene, and n′ isthe variety of unique guide RNAs identified in step (1) that target thegene. The third step comprises calculating average enrichment scores forthe guide RNAs identified in step (1). The enrichment score for a guideRNA is the relative abundance of the guide RNA in theaggregation-negative population of cells divided by the relativeabundance of the guide RNA in the aggregation-positive cells or thepopulation of cells cultured after introduction of the guide RNA libraryor the seeded population of cells. The relative abundance is the readcount of the guide RNA divided by the read count of the total populationof the plurality of unique guide RNAs. The fourth step comprisesselecting the gene if a guide RNA targeting the gene is significantlybelow the random chance of being present and above a thresholdenrichment score. Possible threshold enrichment scores are discussedabove. As a specific example, the threshold enrichment score can be setat about 1.5-fold.

Alternatively, in some CRISPRn methods, the following steps are taken toidentify a gene as a genetic modifier of tau aggregation, whereindisruption of the gene promotes or enhances (or is expected to promoteor enhance) tau aggregation. Likewise, in some CRISPRa methods, thefollowing steps are taken to identify a gene as a genetic modifier oftau aggregation, wherein transcriptional activation of the gene promotesor enhances (or is expected to promote or enhance) tau aggregation. Thefirst step comprises identifying which of the plurality of unique guideRNAs are present in the aggregation-positive population of cells. Thesecond step comprises calculating the random chance of the guide RNAsidentified being present using the formula nCn′*(x−n′)C(m−n)/xCm, wherex is the variety of unique guide RNAs introduced into the population ofcells, m is the variety of unique guide RNAs identified in step (1), nis the variety of unique guide RNAs introduced into the population ofcells that target the gene, and n′ is the variety of unique guide RNAsidentified in step (1) that target the gene. The third step comprisescalculating average enrichment scores for the guide RNAs identified instep (1). The enrichment score for a guide RNA is the relative abundanceof the guide RNA in the aggregation-positive population of cells dividedby the relative abundance of the guide RNA in the aggregation-negativecells or the population of cells cultured after introduction of theguide RNA library or the seeded population of cells. The relativeabundance is the read count of the guide RNA divided by the read countof the total population of the plurality of unique guide RNAs. Thefourth step comprises selecting the gene if a guide RNA targeting thegene is significantly below the random chance of being present and abovea threshold enrichment score. Possible threshold enrichment scores arediscussed above. As a specific example, the threshold enrichment scorecan be set at about 1.5-fold.

Variety when used in the phrase variety of unique guide RNAs means thenumber of unique guide RNA sequences. It is not the abundance, butrather the qualitative “present” or “not present.” Variety of uniqueguide RNAs means the number of unique guide RNA sequence. The variety ofunique guide RNA is determined by next generation sequencing (NGS) toidentify all the unique guide RNAs present in a cell population. It isdone by using two primers that recognize the constant regions of theviral vector to amplify the gRNA that is between the constant regionsand a primer that recognizes one constant region for sequencing. Eachunique guide RNA present in the sample will generate read counts usingthe sequencing primer. The NGS results will include the sequence andalso the number of reads corresponding to the sequence. The number ofreads will be used for the enrichment score calculation for each guideRNA, and the presence of each unique sequence will tell us which guideRNAs are present. For instance, if there are three unique guide RNAs fora gene before selection, and all three are retained post-selection, thenboth n and n′ are 3. These numbers are used for calculating thestatistics but not the actual read counts. However, the read counts foreach guide RNA (in one example, 100, 200, 50, which correspond to eachof the 3 unique guide RNAs) will be used for the calculation ofenrichment score.

VI. Methods of Screening for Genetic Modifiers of Tau Disaggregation

The Cas/tau biosensor cell lines disclosed herein can be used in methodsof screening for genetic modifiers of tau disaggregation (e.g., thatpromote tau disaggregation). Such methods can comprise providing apopulation of tau-aggregation-positive Cas/tau biosensor cells asdisclosed elsewhere herein, introducing a library comprising a pluralityof unique guide RNAs, and assessing tau disaggregation in the targetedcells.

As one example, a method can comprise providing a population of Cas/taubiosensor cells (e.g., a population of cells comprising a Cas protein, afirst tau repeat domain linked to a first reporter, and a second taurepeat domain linked to a second reporter), wherein the cells aretau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state, introducing into the population ofcells a library comprising a plurality of unique guide RNAs that targeta plurality of genes, and culturing the population of cells to allowgenome editing and expansion. The plurality of unique guide RNAs formcomplexes with the Cas protein, and the Cas protein cleaves theplurality of genes resulting in knockout of gene function to produce agenetically modified population of cells. The culturing results in anaggregation-positive population of cells and an aggregation-negativepopulation of cells, which can then be identified. Finally, abundance ofeach of the plurality of unique guide RNAs can be determined in theaggregation-positive population of cells relative to theaggregation-negative population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points. Enrichment of a guide RNA in theaggregation-negative population of cells relative to theaggregation-positive population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau disaggregation, whereindisruption of the gene targeted by the guide RNA promotes taudisaggregation, or is a candidate genetic modifier of tau disaggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to promote taudisaggregation. Likewise, depletion of a guide RNA in theaggregation-positive population of cells relative to theaggregation-negative population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau disaggregation, whereindisruption of the gene targeted by the guide RNA promotes taudisaggregation, or is a candidate genetic modifier of tau disaggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to promote taudisaggregation. Enrichment of a guide RNA in the aggregation-positivepopulation of cells relative to the aggregation-negative population ofcells and/or relative to the population of cells being cultured afterintroduction of the guide RNA library at one or more time pointsindicates that the gene targeted by the guide RNA is a genetic modifierof tau aggregation, wherein disruption of the gene targeted by the guideRNA promotes or enhances tau aggregation, or is a candidate geneticmodifier of tau aggregation (e.g., for further testing via secondaryscreens), wherein disruption of the gene targeted by the guide RNA isexpected to promote or enhance tau aggregation. Likewise, depletion of aguide RNA in the aggregation-negative population of cells relative tothe aggregation-positive population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau aggregation, whereindisruption of the gene targeted by the guide RNA promotes or enhancestau aggregation, or is a candidate genetic modifier of tau aggregation(e.g., for further testing via secondary screens), wherein disruption ofthe gene targeted by the guide RNA is expected to promote or enhance tauaggregation.

Similarly, the SAM/tau biosensor cell lines disclosed herein can be usedin methods of screening for genetic modifiers of tau disaggregation(e.g., that promote tau disaggregation). Such methods can compriseproviding a population of tau-aggregation-positive SAM/tau biosensorcells as disclosed elsewhere herein, introducing a library comprising aplurality of unique guide RNAs, and assessing tau disaggregation in thetargeted cells.

As one example, a method can comprise providing a population of SAM/taubiosensor cells (e.g., a population of cells comprising a chimeric Casprotein comprising a nuclease-inactive Cas protein fused to one or moretranscriptional activation domains, a chimeric adaptor proteincomprising an adaptor protein fused to one or more transcriptionalactivation domains, a first tau repeat domain linked to a firstreporter, and a second tau repeat domain linked to a second reporter),wherein the cells are tau-aggregation-positive cells in which a taurepeat domain stably presents in an aggregated state, introducing intothe population of cells a library comprising a plurality of unique guideRNAs that target a plurality of genes, and culturing the population ofcells to allow transcriptional activation and expansion. The pluralityof unique guide RNAs form complexes with the chimeric Cas protein andthe chimeric adaptor protein, and the complexes activate transcriptionof the plurality of genes resulting in increased gene expression and amodified population of cells. The culturing results in anaggregation-positive population of cells and an aggregation-negativepopulation of cells, which can then be identified. Finally, abundance ofeach of the plurality of unique guide RNAs can be determined in theaggregation-positive population of cells relative to theaggregation-negative population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points. Enrichment of a guide RNA in theaggregation-negative population of cells relative to theaggregation-positive population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau disaggregation, whereintranscriptional activation of the gene targeted by the guide RNApromotes tau disaggregation, or is a candidate genetic modifier of taudisaggregation (e.g., for further testing via secondary screens),wherein transcriptional activation of the gene targeted by the guide RNAis expected to promote tau disaggregation. Likewise, depletion of aguide RNA in the aggregation-positive population of cells relative tothe aggregation-negative population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau disaggregation, whereintranscriptional activation of the gene targeted by the guide RNApromotes tau disaggregation, or is a candidate genetic modifier of taudisaggregation (e.g., for further testing via secondary screens),wherein transcriptional activation of the gene targeted by the guide RNAis expected to promote tau disaggregation. Enrichment of a guide RNA inthe aggregation-positive population of cells relative to theaggregation-negative population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau aggregation, whereintranscriptional activation of the gene targeted by the guide RNApromotes or enhances tau aggregation, or is a candidate genetic modifierof tau aggregation (e.g., for further testing via secondary screens),wherein transcriptional activation of the gene targeted by the guide RNAis expected to promote or enhance tau aggregation. Likewise, depletionof a guide RNA in the aggregation-negative population of cells relativeto the aggregation-positive population of cells and/or relative to thepopulation of cells being cultured after introduction of the guide RNAlibrary at one or more time points indicates that the gene targeted bythe guide RNA is a genetic modifier of tau aggregation, whereintranscriptional activation of the gene targeted by the guide RNApromotes or enhances tau aggregation, or is a candidate genetic modifierof tau aggregation (e.g., for further testing via secondary screens),wherein transcriptional activation of the gene targeted by the guide RNAis expected to promote or enhance tau aggregation.

The Cas/tau biosensor cells used in the method can be any of the Cas/taubiosensor cells disclosed elsewhere herein. Likewise, the SAM/taubiosensor cells used in the method can be any of the SAM/tau biosensorcells disclosed elsewhere herein. The first tau repeat domain and thesecond tau repeat domain can be different or can be similar or the same.The tau repeat domain can be any of the tau repeat domains disclosedelsewhere herein. For example, the first tau repeat domain and/or thesecond tau repeat domain can be a wild type tau repeat domain or cancomprise a pro-aggregation mutation (e.g., a pathogenic, pro-aggregationmutation), such as a tau P301S mutation. The first tau repeat domainand/or the second tau repeat domain can comprise a tau four-repeatdomain. As one specific example, the first tau repeat domain and/or thesecond tau repeat domain can comprise, consist essentially of, orconsist of SEQ ID NO: 11 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In onespecific example, the nucleic acid encoding the tau repeat domain cancomprise, consist essentially of, or consist of SEQ ID NO: 12 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 12, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 11.

The first tau repeat domain can be linked to the first reporter and thesecond tau repeat domain can be linked to the second reporter by anymeans. For example, the reporter can be fused to the tau repeat domain(e.g., as part of a fusion protein). The reporter proteins can be anypair of reporter proteins that produce a detectable signal when thefirst tau repeat domain linked to the first reporter is aggregated withthe second tau repeat domain linked to the second reporter. As oneexample, the first and second reporters can be a split luciferaseprotein. As another example, the first and second reporter proteins canbe a fluorescence resonance energy transfer (FRET) pair. FRET is aphysical phenomenon in which a donor fluorophore in its excited statenon-radiatively transfers its excitation energy to a neighboringacceptor fluorophore, thereby causing the acceptor to emit itscharacteristic fluorescence. Examples of FRET pairs (donor and acceptorfluorophores) are well known. See, e.g., Bajar et al. (2016) Sensors(Basel) 16(9):1488, herein incorporated by reference in its entirety forall purposes. As one specific example of a FRET pair, the first reportercan be cyan fluorescent protein (CFP) and the second reporter can beyellow fluorescent protein (YFP). As a specific example, the CFP cancomprise, consist essentially of, or consist of SEQ ID NO: 13 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 13. As another specific example, the YFP cancomprise, consist essentially of, or consist of SEQ ID NO: 15 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 15.

For the Cas/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the Cas protein cancomprise, consist essentially of, or consist of SEQ ID NO: 21 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 21.

One or more or all of the Cas protein, the first tau repeat domainlinked to the first reporter, and the second tau repeat domain linked tothe second reporter can be stably expressed in the population of cells.For example, nucleic acids encoding one or more or all of the Casprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can begenomically integrated in the population of cells. In one specificexample, the nucleic acid encoding the Cas protein can comprise, consistessentially of, or consist of SEQ ID NO: 22 or a sequence at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNO: 22, optionally wherein the nucleic acid encodes a proteincomprising, consisting essentially of, or consisting of SEQ ID NO: 21.

For the SAM/tau biosensor cells, the Cas protein can be any Cas proteindisclosed elsewhere herein. As one example, the Cas protein can be aCas9 protein. For example, the Cas9 protein can be a Streptococcuspyogenes Cas9 protein. As one specific example, the chimeric Cas proteincan comprise the nuclease-inactive Cas protein fused to a VP64transcriptional activation domain. For example, the chimeric Cas proteincan comprise from N-terminus to C-terminus: the nuclease-inactive Casprotein; a nuclear localization signal; and the VP64 transcriptionalactivator domain. As one specific example, the adaptor protein can be anMS2 coat protein, and the one or more transcriptional activation domainsin the chimeric adaptor protein can comprise a p65 transcriptionalactivation domain and an HSF1 transcriptional activation domain. Forexample, the chimeric adaptor protein can comprise from N-terminus toC-terminus: the MS2 coat protein; a nuclear localization signal; the p65transcriptional activation domain; and the HSF1 transcriptionalactivation domain. In one specific example, the nucleic acid encodingthe chimeric Cas protein can comprise, consist essentially of, orconsist of SEQ ID NO: 38 or a sequence at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38,optionally wherein the nucleic acid encodes a protein comprising,consisting essentially of, or consisting of SEQ ID NO: 36. In onespecific example, the nucleic acid encoding the chimeric adaptor proteincan comprise, consist essentially of, or consist of SEQ ID NO: 39 or asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NO: 39, optionally wherein the nucleic acidencodes a protein comprising, consisting essentially of, or consistingof SEQ ID NO: 37.

One or more or all of the chimeric Cas protein, the chimeric adaptorprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter can be stablyexpressed in the population of cells. For example, nucleic acidsencoding one or more or all of the chimeric Cas protein, the chimericadaptor protein, the first tau repeat domain linked to the firstreporter, and the second tau repeat domain linked to the second reportercan be genomically integrated in the population of cells.

As disclosed elsewhere herein, the cells can be any type of cells. Forexample, the cells can be eukaryotic cells, mammalian cells, or humancells (e.g., HEK293T cells or neuronal cells).

The plurality of unique guide RNAs can be introduced into the populationof cells by any known means. In some methods, the guide RNAs areintroduced into the population of cells by viral transduction, such asretroviral, adenoviral, or lentiviral transduction. In a specificexample, the guide RNAs can be introduced by lentiviral transduction.Each of the plurality of unique guide RNAs can be in a separate viralvector. The population of cells can be infected at any multiplicity ofinfection. For example, the multiplicity of infection can be betweenabout 0.1 and about 1.0, between about 0.1 and about 0.9, between about0.1 and about 0.8, between about 0.1 and about 0.7, between about 0.1and about 0.6, between about 0.1 and about 0.5, between about 0.1 andabout 0.4, or between about 0.1 and about 0.3. Alternatively, themultiplicity of infection can be less than about 1.0, less than about0.9, less than about 0.8, less than about 0.7, less than about 0.6, lessthan about 0.5, less than about 0.4, less than about 0.3, or less thanabout 0.2. In a specific example, the multiplicity of infection can beless than about 0.3.

The guide RNAs can be introduced into the population of cells togetherwith a selection marker or reporter gene to select for cells that havethe guide RNAs, and the method can further comprise selecting cells thatcomprise the selection marker or reporter gene. Examples of selectionmarkers and reporter genes are provided elsewhere herein. As oneexample, the selection marker can be one that imparts resistance to adrug, such as neomycin phosphotransferase, hygromycin Bphosphotransferase, puromycin-N-acetyltransferase, and blasticidin Sdeaminase. Another exemplary selection marker is bleomycin resistanceprotein, encoded by the Sh ble gene (Streptoalloteichus hindustanusbleomycin gene), which confers zeocin (phleomycin D1) resistance. Forexample, cells can be selected with a drug (e.g., puromycin) so thatonly cells transduced with a guide RNA construct are preserved for beingused to carry out screening. For example, the drug can be puromycin orzeocin (phleomycin D1).

In some methods, the plurality of unique guide RNAs are introduced at aconcentration selected such that a majority of the cells receive onlyone of the unique guide RNAs. For example, if the guide RNAs are beingintroduced by viral transduction, the cells can be infected at a lowmultiplicity of infection to ensure that most cells receive only oneviral construct with high probability. As one specific example, themultiplicity of infection can be less than about 0.3.

The population of cells into which the plurality of unique guide RNAs isintroduced can be any suitable number of cells. For example, thepopulation of cells can comprise greater than about 50, greater thanabout 100, greater than about 200, greater than about 300, greater thanabout 400, greater than about 500, greater than about 600, greater thanabout 700, greater than about 800, greater than about 900, or greaterthan about 1000 cells per unique guide RNA. In a specific example, thepopulation of cells comprises greater than about 300 cells or greaterthan about 500 cells per unique guide RNA.

The plurality of unique guide RNAs can target any number of genes. Forexample, the plurality of unique guide RNAs can target about 50 or moregenes, about 100 or more genes, about 200 or more genes, about 300 ormore genes, about 400 or more genes, about 500 or more genes, about 1000or more genes, about 2000 or more genes, about 3000 or more genes, about4000 or more genes, about 5000 or more genes, about 10000 or more genes,or about 20000 or more genes. In some methods, the guide RNAs can beselected to target genes in a particular signaling pathway. In somemethods, the library of unique guide RNAs is a genome-wide library.

The plurality of unique guide RNAs can target any number of sequenceswithin each individual targeted gene. In some methods, a plurality oftarget sequences are targeted on average in each of the targetedplurality of genes. For example, about 2 to about 10, about 2 to about9, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2to about 5, about 2 to about 4, or about 2 to about 3 unique targetsequences can be targeted on average in each of the targeted pluralityof genes. For example, at least about 2, at least about 3, at leastabout 4, at least about 5, or at least about 6 unique target sequencescan be targeted on average in each of the targeted plurality of genes.As a specific example, about 6 target sequences can be targeted onaverage in each of the targeted plurality of genes. As another specificexample, about 3 to about 6 or about 4 to about 6 target sequences aretargeted on average in each of the targeted plurality of genes.

The guide RNAs can target any desired location in the target genes. Insome CRISPRn methods using the Cas/tau biosensor cells, each guide RNAtargets a constitutive exon if possible. In some methods, each guide RNAtargets a 5′ constitutive exon if possible. In some methods, each guideRNA targets a first exon, a second exon, or a third exon (from the 5′end of the gene) if possible. In some CRISPRa methods using the SAM/taubiosensor cells, each guide RNA can target a guide RNA target sequencewithin 200 bp upstream of a transcription start site, if possible. Insome CRISPRa methods using the SAM/tau biosensor cells, wherein eachguide RNA can comprise one or more adaptor-binding elements to which thechimeric adaptor protein can specifically bind. In one example, eachguide RNA comprises two adaptor-binding elements to which the chimericadaptor protein can specifically bind, optionally wherein a firstadaptor-binding element is within a first loop of each of the one ormore guide RNAs, and a second adaptor-binding element is within a secondloop of each of the one or more guide RNAs. For example, theadaptor-binding element can comprise the sequence set forth in SEQ IDNO: 33. In a specific example, each of one or more guide RNAs is asingle guide RNA comprising a CRISPR RNA (crRNA) portion fused to atransactivating CRISPR RNA (tracrRNA) portion, and the first loop is thetetraloop corresponding to residues 13-16 of SEQ ID NO: 17, 19, 30, or31, and the second loop is the stem loop 2 corresponding to residues53-56 of SEQ ID NO: 17, 19, 30, or 31.

The step of culturing the population of cells to allow genome editingand expansion can be any suitable period of time. For example, theculturing can be for between about 2 days and about 15 days, betweenabout 3 days and about 14 days, between about 5 days and about 14 days,between about 7 days and about 14 days, between about 9 days and about14 days, between about 10 days and about 14 days, between about 11 daysand about 14 days, or between about 12 days and about 14 days. Likewise,the step of culturing the population of cells to allow transcriptionalactivation and expansion can be any suitable period of time. Forexample, the culturing can be for between about 2 days and about 15days, between about 3 days and about 14 days, between about 5 days andabout 14 days, between about 7 days and about 14 days, between about 9days and about 14 days, between about 10 days and about 14 days, betweenabout 11 days and about 14 days, or between about 12 days and about 14days.

The step of identifying the aggregation-positive cell population and theaggregation-negative cell population can comprise synchronizing cellcycle progression. For example, this can be used to obtain a cellpopulation predominantly enriched in the S phase or predominantlyenriched in the G2 phase. As a specific example, the synchronization canbe achieved by double thymidine block.

Aggregation can be determined by any suitable means, depending on thereporters used. For example, in methods in which the first reporter andthe second reporter are a fluorescence resonance energy transfer (FRET)pair, the aggregation-positive population of cells can be identified byflow cytometry.

Abundance of guide RNAs can be determined by any suitable means. In aspecific example, abundance is determined by next-generation sequencing.Next-generation sequencing refers to non-Sanger-based high-throughputDNA sequencing technologies. For example, determining abundance of aguide RNA can comprise measuring read counts of the guide RNA.

In some methods, a guide RNA is considered enriched inaggregation-negative cells if the abundance of the guide RNA relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-negative population of cells relativeto the aggregation-positive population of cells and/or the culturedpopulation of cells at one or more time points. In some methods, a guideRNA is considered depleted in aggregation-positive cells if theabundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold lower in theaggregation-positive population of cells relative to theaggregation-negative population of cells and/or the cultured populationof cells at one or more time points. Different enrichment/depletionthresholds can also be used. For example, an enrichment/depletionthreshold can be set higher to be more stringent (e.g., at least about1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at leastabout 1.9-fold, at least about 2-fold, at least about 2.5-fold, or atleast about 2.5-fold). Alternatively, an enrichment/depletion thresholdcan be set lower to be less stringent (e.g., at least about 1.4-fold, atleast about 1.3-fold, or at least about 1.2-fold).

In some methods, a guide RNA is considered enriched inaggregation-positive cells if the abundance of the guide RNA relative tothe total population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-positive population of cells relativeto the aggregation-negative population of cells and/or the culturedpopulation of cells at one or more time points. In some methods, a guideRNA is considered depleted in aggregation-negative cells if theabundance of the guide RNA relative to the total population of theplurality of unique guide RNAs is at least 1.5-fold lower in theaggregation-negative population of cells relative to theaggregation-positive population of cells and/or the cultured populationof cells at one or more time points. Different enrichment/depletionthresholds can also be used. For example, an enrichment/depletionthreshold can be set higher to be more stringent (e.g., at least about1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at leastabout 1.9-fold, at least about 2-fold, at least about 2.5-fold, or atleast about 2.5-fold). Alternatively, an enrichment/depletion thresholdcan be set lower to be less stringent (e.g., at least about 1.4-fold, atleast about 1.3-fold, or at least about 1.2-fold).

In one example, the step of determining abundance can comprisedetermining abundance of the plurality of unique guide RNAs in theaggregation-positive population relative to the aggregation-negativepopulation and/or relative to the population of cells cultured afterintroduction of the guide RNA library at a first time point and/orrelative to the population of cells cultured after introduction of theguide RNA library at a second time point. Likewise, the step ofdetermining abundance can comprise determining abundance of theplurality of unique guide RNAs in the aggregation-negative populationrelative to the aggregation-positive population and/or relative to thepopulation of cells cultured after introduction of the guide RNA libraryat a first time point and/or relative to the population of cellscultured after introduction of the guide RNA library at a second timepoint. For example, the first time point can be at a first passage ofculturing the population of cells or in the middle of culturing thepopulation of cells to allow genome editing and expansion, and thesecond time point can be in the middle of culturing the population ofcells to allow genome editing and expansion. For example, the first timepoint can be after a sufficient amount of time for the guide RNAs toform complexes with the Cas protein, and for the Cas protein to cleavethe plurality of genes resulting in knockout of gene function (CRISPRn)or to transcriptionally activate the plurality of genes (CRISPRa).However, in some cases, the first time point should ideally be at thefirst cell passage to determine the gRNA library representation soonafter infection (i.e., before further expansion and genome editing) andto determine if each gRNA representation evolves from the first timepoint to the second time points and to any additional time points to afinal time point. This allows ruling out enriched gRNAs/targets due tocell growth advantages during the course of the screen by verifying gRNAabundance is unchanged between the first and second time points. As aspecific example, the first time point can be after about 1 day, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 7days, or about 8 days of culturing and expansion (e.g., at about 7 daysof culture and expansion), and the second time point can be after about5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10days, about 11 days, or about 12 days (e.g., about 10 days) of culturingand expansion. For example, the first time point can be after about 7days of culturing and expansion, and the second time point can be afterabout 10 days of culturing and expansion.

In some methods, a gene can then be considered a genetic modifier of taudisaggregation, wherein disruption (CRISPRn) or transcriptionalactivation (CRISPRa) of the gene promotes (or is expected to promote)tau disaggregation, if the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold higher in the aggregation-negative population ofcells relative to the aggregation-positive population of cells, thecultured population of cells at the first time point, and the culturedpopulation of cells at the second time point. Alternatively oradditionally, a gene can then be considered a genetic modifier of taudisaggregation, wherein disruption (CRISPRn) or transcriptionalactivation (CRISPRa) of the gene promotes (or is expected to promote)tau disaggregation, if the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold higher in the aggregation-negative population ofcells relative to the aggregation-positive population of cells and thecultured population of cells at the second time point. Alternatively oradditionally, a gene can then be considered a genetic modifier of taudisaggregation, wherein disruption (CRISPRn) or transcriptionalactivation (CRISPRa) of the gene promotes (or is expected to promote)tau disaggregation, if the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-positive population ofcells relative to the aggregation-negative population of cells, thecultured population of cells at the first time point, and the culturedpopulation of cells at the second time point. Alternatively oradditionally, a gene can then be considered a genetic modifier of taudisaggregation, wherein disruption (CRISPRn) or transcriptionalactivation (CRISPRa) of the gene promotes (or is expected to promote)tau disaggregation, if the abundance of a guide RNA targeting the generelative to the total population of the plurality of unique guide RNAsis at least 1.5-fold lower in the aggregation-positive population ofcells relative to the aggregation-negative population of cells and thecultured population of cells at the second time point.

In some methods, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene promotes or enhances (or is expected to promote orenhance) tau aggregation, if the abundance of a guide RNA targeting thegene relative to the total population of the plurality of unique guideRNAs is at least 1.5-fold higher in the aggregation-positive populationof cells relative to the aggregation-negative population of cells, thecultured population of cells at the first time point, and the culturedpopulation of cells at the second time point. Alternatively oradditionally, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene promotes or enhances (or is expected to promote orenhance) tau aggregation, if the abundance of a guide RNA targeting thegene relative to the total population of the plurality of unique guideRNAs is at least 1.5-fold higher in the aggregation-positive populationof cells relative to the aggregation-negative population of cells andthe cultured population of cells at the second time point. Alternativelyor additionally, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene promotes or enhances (or is expected to promote orenhance) tau aggregation, if the abundance of a guide RNA targeting thegene relative to the total population of the plurality of unique guideRNAs is at least 1.5-fold lower in the aggregation-negative populationof cells relative to the aggregation-positive population of cells, thecultured population of cells at the first time point, and the culturedpopulation of cells at the second time point. Alternatively oradditionally, a gene can then be considered a genetic modifier of tauaggregation, wherein disruption (CRISPRn) or transcriptional activation(CRISPRa) of the gene promotes or enhances (or is expected to promote orenhance) tau aggregation, if the abundance of a guide RNA targeting thegene relative to the total population of the plurality of unique guideRNAs is at least 1.5-fold lower in the aggregation-negative populationof cells relative to the aggregation-positive population of cells andthe cultured population of cells at the second time point.

In some CRISPRn methods, the following steps are taken to identify agene as a genetic modifier of tau disaggregation, wherein disruption ofthe gene promotes (or is expected to promote) tau disaggregation.Likewise, in some CRISPRa methods, the following steps are taken toidentify a gene as a genetic modifier of tau disaggregation, whereintranscriptional activation of the gene promotes (or is expected topromote) tau disaggregation. The first step comprises identifying whichof the plurality of unique guide RNAs are present in theaggregation-negative population of cells. The second step comprisescalculating the random chance of the guide RNAs identified being presentusing the formula nCn′*(x−n′)C(m−n)/xCm, where x is the variety ofunique guide RNAs introduced into the population of cells, m is thevariety of unique guide RNAs identified in step (1), n is the variety ofunique guide RNAs introduced into the population of cells that targetthe gene, and n′ is the variety of unique guide RNAs identified in step(1) that target the gene. The third step comprises calculating averageenrichment scores for the guide RNAs identified in step (1). Theenrichment score for a guide RNA is the relative abundance of the guideRNA in the aggregation-negative population of cells divided by therelative abundance of the guide RNA in the aggregation-positive cells orthe population of cells cultured after introduction of the guide RNAlibrary at the first or second time point. The relative abundance is theread count of the guide RNA divided by the read count of the totalpopulation of the plurality of unique guide RNAs. The fourth stepcomprises selecting the gene if a guide RNA targeting the gene issignificantly below the random chance of being present and above athreshold enrichment score. Possible threshold enrichment scores arediscussed above. As a specific example, the threshold enrichment scorecan be set at about 1.5-fold.

In some CRISPRn methods, the following steps are taken to identify agene as a genetic modifier of tau aggregation, wherein disruption of thegene promotes or enhances (or is expected to promote or enhance) tauaggregation. Likewise, in some CRISPRa methods, the following steps aretaken to identify a gene as a genetic modifier of tau aggregation,wherein transcriptional activation of the gene promotes or enhances (oris expected to promote or enhance) tau aggregation. The first stepcomprises identifying which of the plurality of unique guide RNAs arepresent in the aggregation-positive population of cells. The second stepcomprises calculating the random chance of the guide RNAs identifiedbeing present using the formula nCn′*(x−n′)C(m−n)/xCm, where x is thevariety of unique guide RNAs introduced into the population of cells, mis the variety of unique guide RNAs identified in step (1), n is thevariety of unique guide RNAs introduced into the population of cellsthat target the gene, and n′ is the variety of unique guide RNAsidentified in step (1) that target the gene. The third step comprisescalculating average enrichment scores for the guide RNAs identified instep (1). The enrichment score for a guide RNA is the relative abundanceof the guide RNA in the aggregation-positive population of cells dividedby the relative abundance of the guide RNA in the aggregation-negativecells or the population of cells cultured after introduction of theguide RNA library at the first or second time point. The relativeabundance is the read count of the guide RNA divided by the read countof the total population of the plurality of unique guide RNAs. Thefourth step comprises selecting the gene if a guide RNA targeting thegene is significantly below the random chance of being present and abovea threshold enrichment score. Possible threshold enrichment scores arediscussed above. As a specific example, the threshold enrichment scorecan be set at about 1.5-fold.

Variety when used in the phrase variety of unique guide RNAs means thenumber of unique guide RNA sequences. It is not the abundance, butrather the qualitative “present” or “not present.” Variety of uniqueguide RNAs means the number of unique guide RNA sequence. The variety ofunique guide RNA is determined by next generation sequencing (NGS) toidentify all the unique guide RNAs present in a cell population. It isdone by using two primers that recognize the constant regions of theviral vector to amplify the gRNA that is between the constant regionsand a primer that recognizes one constant region for sequencing. Eachunique guide RNA present in the sample will generate read counts usingthe sequencing primer. The NGS results will include the sequence andalso the number of reads corresponding to the sequence. The number ofreads will be used for the enrichment score calculation for each guideRNA, and the presence of each unique sequence will tell us which guideRNAs are present. For instance, if there are three unique guide RNAs fora gene before selection, and all three are retained post-selection, thenboth n and n′ are 3. These numbers are used for calculating thestatistics but not the actual read counts. However, the read counts foreach guide RNA (in one example, 100, 200, 50, which correspond to eachof the 3 unique guide RNAs) will be used for the calculation ofenrichment score.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleotide sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. When a nucleotide sequence encodingan amino acid sequence is provided, it is understood that codondegenerate variants thereof that encode the same amino acid sequence arealso provided. The amino acid sequences follow the standard conventionof beginning at the amino terminus of the sequence and proceedingforward (i.e., from left to right in each line) to the carboxy terminus.

TABLE 2 Description of Sequences. SEQ ID NO Type Description 1 ProteinTau R1 Repeat Domain 2 Protein Tau R2 Repeat Domain 3 Protein Tau R3Repeat Domain 4 Protein Tau R4 Repeat Domain 5 DNA Tau R1 Repeat DomainCoding Sequence 6 DNA Tau R2 Repeat Domain Coding Sequence 7 DNA Tau R3Repeat Domain Coding Sequence 8 DNA Tau R4 Repeat Domain Coding Sequence9 Protein Tau Four-Repeat Domain (R1-R4; amino acids 243-375 offull-length (P10636-8) Tau) 10 DNA Coding Sequence for Tau Four-RepeatDomain (R1-R4; coding sequence for amino acids 243-375 of full-length(P10636-8) Tau) 11 Protein Tau Four-Repeat Domain (R1-R4) with P301SMutation 12 DNA Coding Sequence for Tau Four-Repeat Domain (R1-R4) withP301S Mutation 13 Protein eCFP 14 DNA eCFP Coding Sequence 15 ProteineYFP 16 DNA eYFP Coding Sequence 17 RNA Guide RNA Scaffold V1 18 RNAGuide RNA Scaffold V2 19 RNA Guide RNA Scaffold V3 20 RNA Guide RNAScaffold V4 21 Protein Cas9-FLAG 22 DNA Cas9-FLAG Coding Sequence 23 RNAcrRNA Tail 24 RNA TracrRNA 25 DNA Guide RNA Target Sequence Plus PAM V126 DNA Guide RNA Target Sequence Plus PAM V2 27 DNA Guide RNA TargetSequence Plus PAM V3 28 RNA TracrRNA v2 29 RNA TracrRNA v3 30 RNA GuideRNA Scaffold V5 31 RNA Guide RNA Scaffold V6 32 RNA Guide RNA ScaffoldV7 33 RNA MS2-binding loop 34 RNA Guide RNA Scaffold with MS2-BindingLoops 35 RNA Generic sgRNA with MS2-Binding Loops 36 Protein dCas9-VP64chimeric Cas protein 37 Protein MCP-p65-HSF1 chimeric adaptor protein 38DNA DNA Encoding dCas9-VP64 chimeric Cas protein 39 DNA DNA EncodingMCP-p65-HSF1 chimeric adaptor protein 40 Protein MCP 41 DNA DNA EncodingMCP 42 DNA Lenti dCas9-VP64 43 DNA Lenti MCP-p65-HSF1_Hygro

EXAMPLES Example 1. Development of Genome-Wide CRISPR/Cas9 ScreeningPlatform to Identify Genetic Modifiers of Tau Aggregation

Abnormal aggregation or fibrillization of proteins is a defining featureof many diseases, notably including a number of neurodegenerativediseases such as Alzheimer's disease (AD), Parkinson's disease (PD),frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS),chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease (CJD),and others. In many of these diseases, the fibrillization of certainproteins into insoluble aggregates is not only a hallmark of disease,but has also been implicated as a causative factor of neurotoxicity.Furthermore, these diseases are characterized by propagation ofaggregate pathology through the central nervous system followingstereotypical patterns, a process which correlates with diseaseprogression. The identification of genes and genetic pathways thatmodify the processes of abnormal protein aggregation, or cell-to-cellpropagation of aggregates, are therefore of great value in betterunderstanding neurodegenerative disease etiology as well as in devisingstrategies for therapeutic intervention.

To identify genes and pathways that modify the processes of abnormal tauprotein aggregation, a platform was developed for performing genome-widescreens with CRISPR nuclease (CRISPRn) sgRNA libraries to identify genesthat regulate the potential of cells to be “seeded” by taudisease-associated protein aggregates (i.e. genes which, when disrupted,cause cells to be more susceptible to tau aggregate formation whenexposed to a source of tau fibrillized protein). The identification ofsuch genes may elucidate the mechanisms of tau cell-to-cell aggregatepropagation and genetic pathways that govern the susceptibility ofneurons to form tau aggregates in the context of neurodegenerativediseases.

The screen employed a tau biosensor human cell line consisting ofHEK293T cells stably expressing tau four-repeat domain, tau_4RD,comprising the tau microtubule binding domain (MBD) with the P301Spathogenic mutation, fused to either CFP or YFP. That is, the HEK293Tcell lines contain two transgenes stably expressing disease-associatedprotein variants fused to the fluorescent protein CFP or the fluorescentprotein YFP: tau^(4RD)-CFP/tau^(4RD)-YFP (TCY), wherein the tau repeatdomain (4RD) comprises the P301S pathogenic mutation. See FIG. 1 . Inthese biosensor lines, tau-CFP/tau-YFP protein aggregation produces aFRET signal, the result of a transfer of fluorescent energy from donorCFP to acceptor YFP. See FIG. 2 . FRET-positive cells, which contain tauaggregates, can be sorted and isolated by flow cytometry. At baseline,unstimulated cells express the reporters in a stable, soluble state withminimal FRET signal. Upon stimulation (e.g., liposome transfection ofseed particles), the reporter proteins form aggregates, producing a FRETsignal. Aggregate-containing cells can be isolated by FACS. Stablypropagating aggregate-containing cell lines, Agg[+], can be isolated byclonal serial dilution of Agg[−] cell lines.

Several modifications were made to this tau biosensor cell line to makeit useful for genetic screening. First, these tau biosensor cells weremodified by introducing a Cas9-expressing transgene (SpCas9) via alentiviral vector. Clonal transgenic cell lines expressing Cas9 wereselected with blasticidin and isolated by clonal serial dilution toobtain single-cell-derived clones. Clones were evaluated for level ofCas9 expression by qRT-PCR (FIG. 3A) and for DNA cleavage activity bydigital PCR (FIG. 3B). Relative Cas9 expression levels are also shown inTable 3.

TABLE 3 Relative Cas9 Expression Levels. Cas9D − Clone Cas9D Ct Cas9DB2m B2m Name rep1 rep2 rep3 rep4 AVG Ct AVG Ct delta Ct 3B5-B1 26.2226.31 26.36 26.45 26.33 22.01 4.33 3B5-G2 23.68 23.85 24.39 23.61 23.8821.51 2.38 7B5-B2 23.63 23.60 24.12 23.50 23.71 21.38 2.34 3B3-A2 24.0523.95 24.02 24.47 24.12 21.94 2.19 7B10-C3 22.58 22.71 22.67 23.20 22.7921.19 1.59 3B5-E1 24.12 24.32 24.75 24.05 24.31 22.81 1.50 3B5-G1 21.1621.14 21.09 21.43 21.20 21.35 −0.15 7B5-C3 19.98 19.99 19.86 19.97 19.9521.24 −1.29 7B5-A2 18.84 18.74 19.33 18.99 18.97 22.10 −3.12 7B5-G119.01 18.88 19.61 19.18 19.17 22.33 −3.16

Specifically, Cas9 mutation efficiency was assessed by digital PCR 3 and7 days after transduction of lentiviruses encoding gRNAs against twoselected target genes. Cutting efficiency was limited by Cas9 levels inlower-expressing clones. A clone with an adequate level of Cas9expression was needed to achieve maximum activity. Several derivedclones with lower Cas9 expression were not able to cut target sequencesefficiently, whereas clones with higher expression (including those usedfor screening) were able to generate mutations at target sequences inthe genes PERK and SNCA with approximately 80% efficiency after threedays in culture. Efficient cutting was observed already at 3 days aftergRNA transduction with only marginal improvement after 7 days. Clone7B10-C3 was selected as a high-performing clone to use for subsequentlibrary screens.

Second, reagents and a method were developed for sensitizing cells totau seeding activity. Tau cell-to-cell propagation may result from tauaggregation activity secreted by aggregate-containing cells. To studycell propagation of tau aggregation, sub-clones were obtained of atau-YFP cell line consisting of HEK293T cells stably expressing taurepeat domain, tau_4RD, comprising the tau microtubule binding domain(MBD) with the P301S pathogenic mutation, fused to YFP. See FIG. 5 .Cells in which tau-YFP protein stably presents in an aggregated state(Agg[+]) were obtained by treating these tau-YFP cells with recombinantfibrillized tau mixed with lipofectamine reagent in order to seed theaggregation of the tau-YFP protein stably expressed by these cells. The“seeded” cells were then serially diluted to obtain single-cell-derivedclones. These clones were then expanded to identify clonal cell lines inwhich tau-YFP aggregates stably persist in all cells with growth andmultiple passages over time. One of these tau-YFP_Agg[+] clones,Clone_18, was used to produce conditioned medium by collecting mediumthat has been on confluent tau-YFP_Agg[+] cells for four days.Conditioned medium (CM) was then applied onto naïve biosensortau-CFP/Tau-YFP cells at a ratio of 3:1 CM:fresh medium so that tauaggregation could be induced in a small percentage of these recipientcells. No lipofectamine was used. Lipofectamine was not used in order tohave an assay that is as physiologic as possible, without tricking therecipient cells to force/increase tau aggregation using lipofectamine.As measured by using flow cytometry to assess the percentage of cellsproducing a FRET signal as a measure of aggregation, conditioned mediumconsistently induced FRET in approximately 0.1% of cells. See FIG. 6 .In conclusion, tau-YFP_Agg[+] cells cannot produce a FRET signal, butthey can provide a source of tau seeds.

Example 2. Genome-Wide CRISPR/Cas9 Screening to Identify GeneticModifiers of Tau Aggregation

To reveal modifier genes of tau aggregation as enriched sgRNAs inFRET[+] cells, the Cas9-expressing tau-CFP/tau-YFP biosensor cellswithout aggregates (Agg[−]) were transduced with two human genome-wideCRISPR sgRNA libraries using a lentiviral delivery approach to introduceknock-out mutations at each target gene. See FIG. 4 . Each CRISPR sgRNAlibrary targets 5′ constitutive exons for functional knock-out with anaverage coverage of ˜3 sgRNAs per gene (total of 6 gRNAs per gene in thetwo libraries combined). Read count distribution (i.e., therepresentation of each gRNA in the library) was normal and similar foreach library. The sgRNAs were designed to avoid off-target effects byavoiding sgRNAs with two or fewer mismatches to off-target genomicsequences. The libraries cover 19,050 human genes and 1864 miRNA with1000 non-targeting control sgRNAs. The libraries were transduced at amultiplicity of infection (MOI) <0.3 at a coverage of >300 cells persgRNA. Tau biosensor cells were grown under puromycin selection toselect cells with integration and expression of a unique sgRNA per cell.Puromycin selection began 24 h after transduction at 1 μg/mL. Fiveindependent screening replicates were used in the primary screen.

Samples of the full, transduced cell population were collected upon cellpassaging at Day 3 and Day 6 post-transduction. After the Day 6 passage,cells were grown in conditioned medium to sensitize them to the seedingactivity. At Day 10, fluorescence-assisted cell sorting (FACS) was usedto isolate specifically the sub-population of FRET[+] cells. See FIG. 7. The screening consisted of five replicated experiments. DNA isolationand PCR amplification of the integrated sgRNA constructs allowed acharacterization by next generation sequencing (NGS) of the sgRNArepertoire at each time point.

Statistical analysis of the NGS data enabled identification of sgRNAsenriched in the Day 10 FRET[+] sub-population of the five experiments ascompared to the sgRNAs repertoire at earlier time points Day 3 and Day6. The concepts of relative abundance and enrichment for NGS analysisare exemplified in FIG. 8 . The first strategy to identify potential taumodifiers was to use DNA sequencing to produce sgRNA read counts in eachsample using the DESeq algorithm to find the sgRNAs that are moreabundant in Day 10 vs. Day 3 or Day 10 vs. Day 6 but not in Day 6 vs.Day 3 (fold change (fc) ≥1.5 and negative binomial test p<0.01). Fc≥1.5means the ratio of (average of day 10 counts)/(average of day 3 or day 6counts)≥1.5. P<0.01 means the chance that there is no statisticaldifference between Day 10 and Day 3 or Day 6 counts <0.01. The DESeqalgorithm is a widely used algorithm for “differential expressionanalysis for sequence count data.” See, e.g., Anders et al. (2010)Genome Biology 11:R106, herein incorporated by reference in its entiretyfor all purposes.

Specifically, two comparisons were used in each library to identify thesignificant sgRNAs: Day 10 vs. Day 3, and Day 10 vs. Day 6. For each ofthese four comparisons, the DESeq algorithm was used, and the cutoffthreshold to be considered as significant was fold change ≥1.5 as wellas negative binomial test p<0.01. Once the significant guides wereidentified in each of these comparisons for each library, a gene wasconsidered to be significant if it meets one of the two followingcriteria: (1) at least two sgRNAs corresponding to the that gene wereconsidered to be significant in one comparison (either Day 10 vs. Day 3or Day 10 vs. Day 6); and (2) at least one sgRNA was significant in bothcomparisons (Day 10 vs. Day 3 and Day 10 vs. Day 6). Using thisalgorithm, we identified five genes to be significant from the firstlibrary and four genes from the second library. See Table 4.

TABLE 4 Genes Identified Using Strategy #1. Day 10 vs Day 3 Day 10 vsDay 6 Day 6 vs Day 3 Significant Significant Significant Gene gRNAs GenegRNAs Gene gRNAs Library #1 Target 1 Target 1 Target 0 Gene 1 Gene 1Gene 1 Target 3 Target 1 Target 0 Gene 2 Gene 2 Gene 2 Target 1 Target 1Target 0 Gene 15 Gene 15 Gene 15 Target 1 Target 1 Target 0 Gene 16 Gene16 Gene 16 Target 2 Target 0 Target 0 Gene 17 Gene 17 Gene 17 Library #2Target 1 Target 1 Target 0 Gene 2 Gene 2 Gene 2 Target 1 Target 1 Target0 Gene 18 Gene 18 Gene 18 Target 1 Target 1 Target 0 Gene 19 Gene 19Gene 19 Target 1 Target 1 Target 0 Gene 20 Gene 20 Gene 20

However, the first strategy requires certain levels of read counthomogeneity within each experiment group might be too stringent. For thesame sgRNA, many factors could produce read count variability among thesamples within each experiment group (Day 3, Day 6 or Day 10 samples),such as initial viral counts in the screening library, infection or geneediting efficiency, and relative growth rate post-gene editing. Thus, asecond strategy was also used based on the positive occurrence (readcount >30) of guides per gene in each sample at Day 10 (post-selection)instead of exact read count. Formal statistical p-value was calculatedfor positively observing a number of guides in the post-selection sample(n′) given the library size (x), number of guides per gene (n), and thetotal number of positive guides in the post-selection sample (m) (the“number” refers to sgRNA type (i.e., unique guide RNA sequences), notread count) (p_(n′)=nCn′*(x−n′)C(m−n)/xCm). The probability of n′ guidesbeing present by chance is: p_(n′)=nCn′*(x−n′)C(m−n)/xCm. Theprobability of n′ guides or more for gene g to be present by chance wascalculated as:p _(g)=Σ_(i=n′) ^(n) p _(i)The overall enrichment of read counts of a gene post-selection comparedto pre-selection was used as additional parameter to identify positivegenes: (Relative abundance=[read count of a gene]/[read count of allgenes] and post-selection enrichment=[relative abundancepost-selection]/[relative abundance pre-selection]).

More specifically, the second strategy is a new and more sensitiveanalysis method for CRISPR positive selection. The goal of CRISPRpositive selection is to use DNA sequencing to identify genes for whichperturbation by sgRNAs is correlated to the phenotype. To reduce thenoise background, multiple sgRNAs for the same gene together withexperiment replicates are usually used in these experiments. However,currently the commonly used statistical analysis methods, which requirea certain degree of homogeneity/agreement among the sgRNAs for the samegene as well as among technical repeats, do not work well. This isbecause these methods cannot handle huge variation among sgRNAs andrepeats for the same gene, due to many possible reasons (e.g., differentinfection or gene editing efficiency, initial viral counts in thescreening library, and the presence of other sgRNAs with the samephenotype). In contrast, we developed a method that is robust to largevariations. It is based on the positive occurrences of guides per genein an individual experiment instead of the exact read count of eachsgRNA. Formal statistical p-values are calculated for positivelyobserving a number of sgRNAs over experiment repeats given the librarysize, number of sgRNAs per gene, and the totally number of positivesgRNAs in each experiment. Relative sgRNA sequence read enrichmentbefore and after phenotype selection is also used as a parameter. Ourmethod performs better than widely used methods up-to-date, includingDESeq, MAGECK, and others. Specifically, this method includes thefollowing steps:

-   -   (1) For each experiment, identifying any present guides in cells        with positive phenotype.    -   (2) At the gene level, calculating the random chance of guides        being present in each experiment: nCn′*(x−n′)C(m−n)/xCm, where x        is the variety of guides before phenotype selection, m is the        variety of guides after phenotype selection, n is the variety of        guides for a gene before phenotype selection, and n′ is the        variety of guides for the gene after phenotype selection. The        overall chance of being present across multiple experiments        (generating a single p value over p values generated from        several experiments) is calculated by the Fisher's combined        probability test (reference: Fisher, R. A.; Fisher, R. A (1948)        “Questions and answers #14” The American Statistician). That is,        a test statistic ϕ is first computed using the p-values from the        multiple experiments: ϕ=−2Σ_(k=1) ^(K)p_(k), where p_(k) is the        p-value calculated for the kth experiment, and K is the total        number of the experiments. Then, the combined p-value over the K        experiments is equal to the probability of observing the value        of ϕ under the chi-square distribution with the degree of        freedom of 2*K. Alternatively, the overall chance of being        present across multiple experiments is calculated by multiplying        the above calculated possibility obtained from each experiment.    -   (3) Calculating the average enrichment of guides at gene level:        Enrichment score=relative abundance post-selection/relative        abundance pre-selection. Relative abundance=read count of guides        for a gene/read count of all guides.    -   (4) Selecting genes significantly below the random chance of        being present as well as above certain enrichment score.

Fourteen of the target genes identified by the two different approaches(either approach or both) as being enriched in the FRET[+] cells wereselected as top candidates for further validation after visualinspection based on read counts data. See Table 5. Thirty individualsgRNAs were tested in secondary screens for validation. A schematic ofthe secondary screens is shown in FIG. 9 , and the results are shown inFIG. 10 . Disruption of either Target Gene 2 or Target Gene 8, bymultiple tested sgRNAs, increased the susceptibility of a cell to formtau aggregates in response to a source of tau seeding activity(conditioned medium). The induction of FRET signal increased by15-20-fold in cells with disruption of either of these two targets. Thedisruption of these two target genes increased the formation of tauaggregates in response to conditioned medium but not fresh medium. SeeFIG. 11 .

TABLE 5 Targets Identified. Target Gene sgRNA Target Gene 1 g1 - Lib-ATarget Gene 1 g5 - Lib-B Target Gene 2 g1 - Lib-A Target Gene 2 g2 -Lib-A Target Gene 2 g3 - Lib-A Target Gene 2 g6 - Lib-B Target Gene 3g2 - Lib-A Target Gene 3 g5 - Lib-B Target Gene 4 g3 - Lib-A Target Gene4 g5 - Lib-B Target Gene 5 g1 - Lib-A Target Gene 5 g4 - Lib-B TargetGene 6 g2 - Lib-A Target Gene 6 g5 - Lib-B Target Gene 7 g1 - Lib-ATarget Gene 7 g5 - Lib-B Target Gene 8 g5 - Lib-B Target Gene 8 g6 -Lib-B Target Gene 9 g2 - Lib-A Target Gene 9 g6 - Lib-B Target Gene 10g1 - Lib-A Target Gene 10 g6 - Lib-B Target Gene 11 g3 - Lib-A TargetGene 11 g4 - Lib-B Target Gene 12 g1 - Lib-A Target Gene 12 g6 - Lib-BTarget Gene 13 g5 - Lib-B Target Gene 13 g6 - Lib-B Target Gene 14 g1 -Lib-A Target Gene 14 g4 - Lib-B

Further experiments with Target Genes 2 and 8 were then performed tofurther validate that targeting of each gene promotes tau aggregation.See FIG. 12 . Two different sgRNAs against Target Gene 2 were tested andone sgRNA against Target Gene 8 were used. A non-targeting sgRNA wasused as a negative control. Four independent lentiviral transductionswere done for each guide RNA on Day 0. On Day 6, tau seeding withconditioned medium was performed with or without lipofectamine andsamples were collected for qRT-PCR. The qRT-PCR data are shown in FIG.13 . Each of the two sgRNAs targeting Target Gene 2 reduced Target Gene2 mRNA expression, and the gRNA targeting Target Gene 8 reduced TargetGene 8 expression. On Day 10, FACS analysis was done to assess inductionof FRET signal. Tau aggregation was increased by each of the two sgRNAstargeting Target Gene 2 and the gRNA targeting Target Gene 8. See FIG.15 . On Day 13, samples were collected for western blot analysis. Thewestern blot results are shown in FIG. 14 . Similar to the qRT-PCRexperiments assessing mRNA expression, expression of Protein 2 wasreduced by the two sgRNAs targeting Target Gene 2, and expression ofProtein 8 was reduced by the sgRNA targeting Target Gene 8.

Further validation of Target Genes 2 and 8 as modifiers of tauaggregation was done by isolating individual Target Gene 2 knockdownclones and individual Target Gene 8 knockdown clones for validation.Cas9-expressing tau-CFP/tau-YFP biosensor cells without aggregates(Agg[−]) were transduced with lentivirus expressing Target Gene 2 sgRNA1, Target Gene 8 sgRNA 5, or a non-targeting sgRNA. Serial clonaldilution was then undertaken to select individual clones. Levels ofTarget Gene 2 mRNA and Target Gene 8 mRNA were assessed by qRT-PCR, andlevels of Protein 2 and Protein 8 were assessed by western blot. EachTarget Gene 2 sgRNA clone had reduced Target Gene 2 mRNA expression(data not shown) and Protein 2 expression (FIG. 16 ), and each targetGene 8 sgRNA clone had reduced Target Gene 8 mRNA expression (data notshown) and Protein 8 expression (FIG. 16 ).

Next, each clone was seeded with conditioned medium for 4 days and FRETanalysis was done to assess tau aggregation. The knockdown clonesvalidate Target Genes 2 and 8 as modifiers of tau aggregation. See FIG.17 .

The individual clones were then further characterized by next-generationsequencing to determine what modifications were made that the TargetGene 2 and Target Gene 8 loci. The modifications are summarized in Table6 below. Almost all of the mutant clones contain some percentage of wildtype alleles. The percentage of FRET[+] cells (tau aggregation activity)correlated with the percentage of insertions/deletions caused bynon-homologous end joining at the cleavage sites (i.e., tau aggregationwas inversely correlated with the percentage of wild type alleles—thelower the percentage of wild type alleles, the higher the percentage ofFret[+] cells). See FIG. 16 and Table 6.

TABLE 6 Characterization of Target Gene 2 and Target Gene 8 Clones. GeneAmplicon Allele Frequency (Reads ≥ 5%) (Target) Clone Sequenced WT INDEL1 INDEL 2 Target MP1-3 TG8_g5 98.80% Gene 2 TG2_g1 11.30% 49.9% (+1 bp)33.9% (Δ16 bp) MP1-10 TG8_g5 98.60% TG2_g1 16.50% 79.1% (+1 bp) MP1-13TG8_g5 98.80% TG2_g1 14.90% 35.9% (Δ6 bp) 44.3% (+1 bp) MP1-23 TG8_g598.70% TG2_g1 20.20% 71.4% (+1 bp) Target MP5-7 TG8_g5 0.00% 54.8% (Δ3bp + 6 bp) 29.7% (C→T) Gene 8 TG2_g1 99.5%% MP5-9 TG8_g5 34.80% 55.0%(Δ20 bp)  6.8% (+1 bp) TG2_g1 99.30%

Tau phosphorylation was also assessed in each clone by western blot. Tauwas found to be hyper-phosphorylated at S262 and S356 in Target Gene 8sgRNA clone 5.7. See FIG. 18 . Although clones in which tau was nothyperphosphorylated still appeared to enhance FRET induction in thisexperiment, clone 5.7 was quite unstable, and Target Gene 8 is involvedin many biological processes, so no general conclusions could be drawn.Further experiments in mouse primary cortical neurons showed thatphosho-tau (S356) staining is increased in the somatodendriticcompartment in mouse primary cortical neurons treated with Cas9 andTarget Gene2 sgRNA via lentiviral delivery and maintained for 14 days inculture (data not shown).

This validation confirmed the value of the primary screening approach inthe identification of genes that can regulate the susceptibility ofcells to tau seeding when exposed to an external source of tau seedingactivity. Targets identified through the screening could be thereforerelevant targets in the cell-to-cell propagation of tau pathology in thecontext of neurodegenerative disease and will be further explored.

Example 3. Development of Genome-Wide CRISPR/Cas9 Screening Platform toIdentify Genetic Modifiers of Tau Aggregation Using a TranscriptionalActivation CRISPR/Cas9 Library

To further identify genes and pathways that modify the processes ofabnormal tau protein aggregation, a platform was developed forperforming genome-wide screens with transcriptional activation (hSAM)CRISPRa sgRNA libraries to identify genes that regulate the potential ofcells to be “seeded” by tau disease-associated protein aggregates (i.e.genes which, when transcriptionally activated, cause cells to be moresusceptible to tau aggregate formation when exposed to a source of taufibrillized protein). The identification of such genes may elucidate themechanisms of tau cell-to-cell aggregate propagation and geneticpathways that govern the susceptibility of neurons to form tauaggregates in the context of neurodegenerative diseases.

The screen employed a tau biosensor human cell line consisting ofHEK293T cells stably expressing tau four-repeat domain, tau_4RD,comprising the tau microtubule binding domain (MBD) with the P301Spathogenic mutation, fused to either CFP or YFP. That is, the HEK293Tcell lines contain two transgenes stably expressing disease-associatedprotein variants fused to the fluorescent protein CFP or the fluorescentprotein YFP: tau^(4RD)-CFP/tau^(4RD)-YFP (TCY), wherein the tau repeatdomain (4RD) comprises the P301S pathogenic mutation. See FIG. 1 . Inthese biosensor lines, tau-CFP/tau-YFP protein aggregation produces aFRET signal, the result of a transfer of fluorescent energy from donorCFP to acceptor YFP. See FIG. 2 . FRET-positive cells, which contain tauaggregates, can be sorted and isolated by flow cytometry. At baseline,unstimulated cells express the reporters in a stable, soluble state withminimal FRET signal. Upon stimulation (e.g., liposome transfection ofseed particles), the reporter proteins form aggregates, producing a FRETsignal. Aggregate-containing cells can be isolated by FACS. Stablypropagating aggregate-containing cell lines, Agg[+], can be isolated byclonal serial dilution of Agg[−] cell lines.

Several modifications were made to this tau biosensor cell line to makeit useful for genetic screening. First, this biosensor cell line wasfurther transgenically modified to express the components of theCRISPR/Cas SAM transcriptional activation system: dCas9-VP64 andMS2-P65-HSF1. Lentiviral dCas9-VP64 and MS2-P65-HSF1 constructs areprovided in SEQ ID NOS: 42 and 43, respectively. A clone was selected asa high-performing clone to use for subsequent library screens. Thisclone was validated for its efficacy in activating selected targetgenes.

Second, reagents and a method were developed for sensitizing cells totau seeding activity as in Example 1. Alternatively, if more aggregationis needed, use of cell lysate from an Agg[+] clone can be used as inExample 5.

Example 4. Genome-Wide CRISPR/Cas9 Screening to Identify GeneticModifiers of Tau Aggregation Using a Transcriptional ActivationCRISPR/Cas9 Library

To reveal further modifier genes of tau aggregation as enriched sgRNAsin FRET[+] cells, the SAM-expressing tau-CFP/tau-YFP biosensor cellswithout aggregates (Agg[−]) were transduced with a human genome-wideCRISPR hSAM sgRNA library using a lentiviral delivery approach totranscriptionally activate each target gene. The sgRNAs in the librarytarget sites within 200 bp upstream of the transcription start site withan average coverage of ˜3 sgRNAs per gene. The sgRNAs were designed toavoid off-target effects by avoiding sgRNAs with two or fewer mismatchesto off-target genomic sequences. The library covers 18,946 human genes.The library was transduced at a multiplicity of infection (MOI) <0.3 ata coverage of >300 cells per sgRNA. Tau biosensor cells were grown underzeocin selection to select cells with integration and expression of aunique sgRNA per cell. Five independent screening replicates were usedin the primary screen.

Samples of the full, transduced cell population were collected upon cellpassaging at Day 3 and Day 6 post-transduction. After the Day 6 passage,cells were grown in conditioned medium to sensitize them to the seedingactivity. At Day 10, fluorescence-assisted cell sorting (FACS) was usedto isolate specifically the sub-population of FRET[+] cells. DNAisolation and PCR amplification of the integrated sgRNA constructsallowed a characterization by next generation sequencing (NGS) of thesgRNA repertoire at each time point.

Because data analysis and statistical analysis mirrored the approachused in Example 2, not all the details from Example 2 are repeated here.Statistical analysis of the NGS data enabled identification of sgRNAsenriched in the Day 10 FRET[+] sub-population of the multipleexperiments as compared to the sgRNAs repertoire at earlier time pointsDay 3 and Day 6. The first strategy to identify potential tau modifierswas to use DNA sequencing to produce sgRNA read counts in each sampleusing the DESeq algorithm to find the sgRNAs that are more abundant inDay 10 vs. Day 3 or Day 10 vs. Day 6 but not in Day 6 vs. Day 3 (foldchange (fc) ≥1.5 and negative binomial test p<0.01). Fc≥1.5 means theratio of (average of day 10 counts)/(average of day 3 or day 6 counts)≥1.5. P<0.01 means the chance that there is no statistical differencebetween Day 10 and Day 3 or Day 6 counts <0.01. The DESeq algorithm is awidely used algorithm for “differential expression analysis for sequencecount data.” See, e.g., Anders et al. (2010) Genome Biology 11:R106,herein incorporated by reference in its entirety for all purposes.

Specifically, two comparisons were used in each library to identify thesignificant sgRNAs: Day 10 vs. Day 3, and Day 10 vs. Day 6. For each ofthese four comparisons, the DESeq algorithm was used, and the cutoffthreshold to be considered as significant was fold change ≥1.5 as wellas negative binomial test p<0.01. Once the significant guides wereidentified in each of these comparisons for each library, a gene wasconsidered to be significant if it meets one of the two followingcriteria: (1) at least two sgRNAs corresponding to the that gene wereconsidered to be significant in one comparison (either Day 10 vs. Day 3or Day 10 vs. Day 6); and (2) at least one sgRNA was significant in bothcomparisons (Day 10 vs. Day 3 and Day 10 vs. Day 6).

However, the first strategy requires certain levels of read counthomogeneity within each experiment group might be too stringent. For thesame sgRNA, many factors could produce read count variability among thesamples within each experiment group (Day 3, Day 6 or Day 10 samples),such as initial viral counts in the screening library, infection or geneediting efficiency, and relative growth rate post-gene editing. Thus, asecond strategy was also used based on the positive occurrence (readcount >30) of guides per gene in each sample at Day 10 (post-selection)instead of exact read count. Formal statistical p-value was calculatedfor positively observing a number of guides in the post-selection sample(n′) given the library size (x), number of guides per gene (n), and thetotal number of positive guides in the post-selection sample (m) (the“number” refers to sgRNA type (i.e., unique guide RNA sequences), notread count) (p_(n′)=nCn′*(x−n′)C(m−n)/xCm). The probability of n′ guidesbeing present by chance is: p_(n′)=nCn′*(x−n′)C(m−n)/xCm. Theprobability of n′ guides or more for gene g to be present by chance wascalculated as:p _(g)=Σ_(i=n′) ^(n) p _(i)The overall enrichment of read counts of a gene post-selection comparedto pre-selection was used as additional parameter to identify positivegenes: (Relative abundance=[read count of a gene]/[read count of allgenes] and post-selection enrichment=[relative abundancepost-selection]/[relative abundance pre-selection]).

More specifically, the second strategy is a new and more sensitiveanalysis method for CRISPR positive selection. The goal of CRISPRpositive selection is to use DNA sequencing to identify genes for whichtranscriptional activation by sgRNAs is correlated to the phenotype. Toreduce the noise background, multiple sgRNAs for the same gene togetherwith experiment replicates are usually used in these experiments.However, currently the commonly used statistical analysis methods, whichrequire a certain degree of homogeneity/agreement among the sgRNAs forthe same gene as well as among technical repeats, do not work well. Thisis because these methods cannot handle huge variation among sgRNAs andrepeats for the same gene, due to many possible reasons (e.g., differentinfection or gene editing efficiency, initial viral counts in thescreening library, and the presence of other sgRNAs with the samephenotype). In contrast, we developed a method that is robust to largevariations. It is based on the positive occurrences of guides per genein an individual experiment instead of the exact read count of eachsgRNA. Formal statistical p-values are calculated for positivelyobserving a number of sgRNAs over experiment repeats given the librarysize, number of sgRNAs per gene, and the totally number of positivesgRNAs in each experiment. Relative sgRNA sequence read enrichmentbefore and after phenotype selection is also used as a parameter. Ourmethod performs better than widely used methods up-to-date, includingDESeq, MAGECK, and others. Specifically, this method includes thefollowing steps:

-   -   (1) For each experiment, identifying any present guides in cells        with positive phenotype.    -   (2) At the gene level, calculating the random chance of guides        being present in each experiment: nCn′*(x−n′)C(m−n)/xCm, where x        is the variety of guides before phenotype selection, m is the        variety of guides after phenotype selection, n is the variety of        guides for a gene before phenotype selection, and n′ is the        variety of guides for the gene after phenotype selection. The        overall chance of being present across multiple experiments        (generating a single p value over p values generated from        several experiments) is calculated by the Fisher's combined        probability test (reference: Fisher, R. A.; Fisher, R. A (1948)        “Questions and answers #14” The American Statistician). That is,        a test statistic ϕ is first computed using the p-values from the        multiple experiments: ϕ=−2Σ_(k=1) ^(K)p_(k), where p_(k) is the        p-value calculated for the kth experiment, and K is the total        number of the experiments. Then, the combined p-value over the K        experiments is equal to the probability of observing the value        of ϕ under the chi-square distribution with the degree of        freedom of 2*K. Alternatively, the overall chance of being        present across multiple experiments is calculated by multiplying        the above calculated possibility obtained from each experiment.    -   (3) Calculating the average enrichment of guides at gene level:        Enrichment score=relative abundance post-selection/relative        abundance pre-selection. Relative abundance=read count of guides        for a gene/read count of all guides.    -   (4) Selecting genes significantly below the random chance of        being present as well as above certain enrichment score.

A total of 34 target genes (targeted by 42 different sgRNAs) wereidentified by the two different approaches (either approach or both) asbeing enriched in the FRET[+] cells.

Example 5. Preparation and Validation of Tau-YFP Clone18 Agg[+] CellLysates for Tau Seeding

We next wanted to identify target genes that, when disrupted, reduce tauaggregation (whether by blocking uptake of seeds, inhibiting theformation of oligomers or fibrils, promoting their disassembly, or bysome other mechanism). To develop such a screen for sgRNAs that preventtau aggregation, we needed a source of seeding activity that is potentand that we can easily generate in large quantities. As described inthis example, we discovered that whole cell lysate from tau-YFP Agg[+]clone18 can induce tau aggregation and FRET signal, while lysate fromtau-YFP Agg[−] does not. Whole cell lysate is much more potent atseeding tau activity (with no lipofectamine) than conditioned media orrecombinant tau fibrils. To induce aggregation (FRET) in largerpercentage of cells (e.g., >50%), we tried using whole cell lysate incombination with lipofectamine. We also needed to develop a method ofcollecting cell lysate in buffer that is not itself toxic to cells. Inthe experiments described herein, we used phosphate-buffered saline(PBS)+protease inhibitors to collect cells and sonication to lyse cells.Cell lysate sonicated for 3 minutes worked best.

Cells were prepared for sonication as follows: (1) take 6×T175 plates oftau-YFP Agg[−] cells and 6×T175 plates of tau-YFP Clone18 Agg[+] cells(˜50 million cells/T175); (2) rinse flask with 10 mL PBS, then scrapecells into 5 mL of PBS; (3) collect 2×T175 into a 15 mL tube, then rinsethese 2×T175 with a total of 5 mL PBS and add it to the 15 mL tube for afinal volume of 15 mL (should have 3 tubes of tau-YFP Agg[−] cells and 3tubes of tau-YFP Clone18 Agg[+] cells); (4) spin the tubes @ 1000 rpmfor 5 minutes, aspirate, add 4 mL PBS/tube+40 L HALT™ (sixbroad-spectrum protease inhibitors AEBSF, aprotinin, bestatin, E-64,leupeptin and pepstatin A stabilized in high-quality dimethylsulfoxide(DMSO))+40 μL ethylenediaminetetracetic acid (EDTA)/tube (proteaseinhibitors); and (5) transfer cell suspensions to 50 mL tubes(sonication tube holder can only hold 50 mL tube) and freeze at −80° C.until sonication.

Sonication to create cell lysates was performed as follows: (1) thawcell suspensions in warm water bath; (2) sonicate for 1 minute, 3minutes, or 6 minutes; (3) spin down sonicated samples @ 1000 rpm for 5minutes, make 300 μL aliquots, and freeze at −80° C.

Tau biosensor cells tau-CFP/tau-YFP/Cas9 Clone 7B10C3 Agg[−] (C3) andFRET/FACS control cells tau-CFP, tau-YFP, HEK-HZ, tau-CFP/tau-YFP/Cas9Clone 7B10C3-B2 Agg[+] (B2) were thawed and were plated in 12-wellplates and were treated with 10 μg, 30 g, or 100 μg of the cell lysateproduced above. The results are shown in FIG. 19 . A dose-dependentresponse was observed, and 3 minutes sonication worked best.

In a subsequent experiment, 150,000 of 7B10C3 Agg[−] cells were platedper well in 24-well plates in duplicate. We used 1 mL of fresh mediumper well. We added 0 (control), 10, 25, or 50 μg of 3-min tau-YFP Agg[+]Clone18 cell lysate+/−lipofectamine (10 μL per 1 mL of medium). With thelipofectamine, all cells were Agg[+], but all cells were dead after 24hours in the 25 and 50 μg lysate samples and after 48 hours in the 10 μglysate samples.

We next tried decreasing the amount of lysate used. 150,000 of 7B10C3Agg[−] cells were plated per well in 24-well plates in duplicate. Weused 1 mL of fresh medium per well. We added 0 (control), 1, 5, or 10 μgof 3-min tau-YFP Agg[+] Clone18 cell lysate+/−lipofectamine (10 μL per 1mL of medium). With the lipofectamine, all cells were Agg[+], but thecells looked unhealthy after 24 hours and were dead after 48 hours inthe 5 and 10 μg lysate samples. In the 1 μg lysate samples, the cellswere all Agg[+], but looked somewhat unhealthy at 24 hours. After 3days, the cells were still alive and Agg[+].

We next tried decreasing the amount of lysate used and testing differentlipofectamine concentrations. 150,000 of 7B10C3 Agg[−] cells were platedper well in 24-well plates in duplicate. We used 1 mL of fresh mediumper well. We added 0 (control), 0.1, 0.5, or 1 jag of 3-min tau-YFPAgg[+] Clone18 cell lysate+/−lipofectamine (1, 5, or 10 μL per 1 mL ofmedium). With 10 μL lipofectamine, all cells were dead after 24 hours inall samples. With 5 μL lipofectamine and 0.5 μg lysate, the cells lookedok after 48 hours and most were Agg[+]. Similarly, with 5 μLlipofectamine and 1 μg lysate, the cells looked ok after 48 hours andall were Agg[+]. However, the 5 μL lipofectamine was toxic in the 0 ugand 0.1 μg samples, as the cells looked unhealthy after 48 hours. With 1μL lipofectamine, the cells looked ok after 48 hours and were ˜30%Agg[+] in the 0.1 μg lysate samples, were ˜40% Agg[+] in the 0.5 μglysate samples, and were ˜50% Agg[+] in the 1 μg lysate samples. SeeFIG. 20 . A similar experiment was done testing 3 μg, 5 μg, or 10 μg ofcell lysate and 1 μL, 2 μL, or 3 μL of lipofectamine. The results areshown in FIG. 21 . Yet another experiment was done testing 0.5 μg, 0.7μg, or 1 μg of cell lysate and 3.5 μL or 4 μL of lipofectamine. Thepercentage of aggregation-positive cells for each condition was asfollows: 0.5 μg lysate/3.5 μL lipofectamine: 73.5%; 0.7 μg lysate/3.5 μLlipofectamine: 71.7%; 1 μg lysate/3.5 μL lipofectamine: 75.7%; 0.5 μglysate/4 μL lipofectamine: 76.4%; 0.7 μg lysate/4 μL lipofectamine:76.7%; and 1 μg lysate/4 μL lipofectamine: 78.0%. In furtherexperiments, 2 μL and 2.5 μL lipofectamine were not toxic to the cells,but 3 μL, 3.5 μL, 4 μL, 4.5 μL, and 5 μL were toxic to cells.

Next, we scaled up to T25 flasks, plating 2.8 million 7B10C3cells+lysate+lipofectamine. FACS/FRET sorting was done after 2 days inculture. Two conditions were tested: (1) 1.5 μg lysate+2 μLlipofectamine per mL of fresh medium; and (2) 2 μg lysate+2 μLlipofectamine per mL of fresh medium. In the first experiment, 965,880cells were FRET[−](i.e., Agg[−]) and 984,760 cells were FRET[+] (i.e.,Agg[+]). In the second experiment, 547,960 cells were FRET[−] (i.e.,Agg[−]) and 855,900 cells were FRET[+] (i.e., Agg[+]). We concluded thatfor genome-wide screens we would use 2.5 μL lipofectamine per mL offresh medium and compare two different doses of tau-YFP Agg[+] clone18cell lysates (1.5 μg and 2 μg), which could potentially increase thepercent of Agg[+] cells while keeping them healthy.

Example 6. Genome-Wide CRISPR/Cas9 Screening to Identify GeneticModifiers that Prevent Tau Aggregation

The goal of the screen in Example 2 was to identify modifier geneswhich, when disrupted, promote the formation of tau aggregates whenstimulated with a weak tau seeding material. In contrast, in the screendescribed in this example, we treated our biosensor cells with a potenttau seeding material that normally causes tau aggregation and FRETinduction in a majority of cells. This “maximal seeding” materialconsists of sonicated whole cell lysate from tau-YFP Agg[+] Clone18cells, applied with lipofectamine transfection reagent. We took thisapproach to identify target genes that, when disrupted, reduce tauaggregation (whether by blocking uptake of seeds, inhibiting theformation of oligomers or fibrils, promoting their disassembly, or bysome other mechanism). See FIG. 22 .

The Cas9-expressing tau-CFP/tau-YFP biosensor cells without aggregates(Agg[−]) were transduced with two human genome-wide CRISPR sgRNAlibraries using a lentiviral delivery approach to introduce knock-outmutations at each target gene. Each CRISPR sgRNA library targets 5′constitutive exons for functional knock-out with an average coverage of˜3 sgRNAs per gene (total of 6 gRNAs per gene in the two librariescombined). Read count distribution (i.e., the representation of eachgRNA in the library) was normal and similar for each library. The sgRNAswere designed to avoid off-target effects by avoiding sgRNAs with two orfewer mismatches to off-target genomic sequences. The libraries cover19,050 human genes and 1864 miRNA with 1000 non-targeting controlsgRNAs. The libraries were transduced at a multiplicity of infection(MOI) <0.3 at a coverage of >300 cells per sgRNA. Tau biosensor cellswere grown under puromycin selection to select cells with integrationand expression of a unique sgRNA per cell. Puromycin selection began 24h after transduction at 1 μg/mL. Five independent screening replicateswere used in the primary screen.

In each replicate, samples of 7B10C3 biosensor cells were collected atDay 3 and Day 7 post-transduction of the lentivirally-packaged CRISPRlibraries. At the Day 7 passage, the “maximal seeding” material wasadded to the cells, and after 48 hours (at Day 9), FACS was performed toseparate and collect FRET[−] and FRET[+] populations. The screeningconsisted of five replicated experiments. DNA isolation and PCRamplification of the integrated sgRNA constructs allowed acharacterization by next generation sequencing (NGS) of the sgRNArepertoire at each time point. There were 40 samples in total: 5replicate screens*2 libraries*4 samples for each (Day 3, Day 7, Day 9FRET[−], and Day 9 FRET[+]). For the “maximal seeding,” 2.5 μL oflipofectamine was used per mL of fresh medium, and different amounts ofcell lysate were tested per mL of fresh medium (2 μg, 4 μg, and 5 μg).

Data analysis and statistical analysis mirrored the approach used inExample 2, so the details from Example 2 are not repeated here. Wecompared (paired analysis) FRET(−) vs FRET(+) vs Day 7. We alsoperformed a paired analysis by comparison to Day 3 (more stringent). Day9 FRET[−] and FRET[+] samples were analyzed for sgRNAs whose enrichmentor depletion may indicate an effect on the tau aggregation phenotype.sgRNAs that are enriched in Day 9 FRET[−] cells relative to Day 9FRET[+], Day 3, and Day 7, and/or are depleted in Day 9 FRET[+] cellsrelative to Day 9 FRET[−], Day 3, and Day 7, indicate target genes that,when disrupted, may reduce or protect against tau aggregation. ThesesgRNA hits are of special interest as potential targets for therapeuticintervention. sgRNAs that are enriched in Day 9 FRET[+] cells relativeto Day 9 FRET[−], Day 3, and Day 7, and/or are depleted in Day 9 FRET[−]cells relative to Day 9 FRET[+], Day 3, and Day 7, indicate target genesthat, when disrupted, may promote or enhance tau aggregation. sgRNAsthat are depleted in both Day 9 FRET[+] and FRET[−] relative to earliertimepoints are likely to represent essential genes that reduce cellviability over time when they are disrupted.

We identified 142 significant gRNAs that were enriched or depleted inFRET[−] cells (as compared to FRET[+] cells and Day 7 cells). Of these,46 gRNAs were depleted in FRET[−] cells, 77 gRNAs were enriched inFRET[−] cells, and 20 gRNAs were enriched in FRET[−] cells compared toDay 7 (not significant as compared to Day 3). See FIGS. 23 and 24 .

Next, 405 individual sgRNAs were tested in secondary screens forvalidation. A schematic of the secondary screens is shown in FIG. 25 .Four experiments were done. The amount of cell lysate used in each was 5μg/mL of fresh medium. Four different amounts of lipofectamine (per mLfresh medium) were tested: 1.5 μL, 2 μL, 2.5 μL, and 3.5 μL. Thisvalidation confirms the value of the primary screening approach in theidentification of genes that can act as positive and negative modifiersof tau aggregation.

Example 7. Genome-Wide CRISPR/Cas9 Screening to Identify GeneticModifiers that Prevent Tau Aggregation Using a TranscriptionalActivation CRISPR/Cas9 Library

Similar to Example 6, in the screen described in this example, wetreated our biosensor cells with a potent tau seeding material thatnormally causes tau aggregation and FRET induction in a majority ofcells. This “maximal seeding” material consists of sonicated whole celllysate from tau-YFP Agg[+] Clone18 cells, applied with lipofectaminetransfection reagent. We took this approach to identify target genesthat, when transcriptionally activated, reduce tau aggregation (whetherby blocking uptake of seeds, inhibiting the formation of oligomers orfibrils, promoting their disassembly, or by some other mechanism). SeeFIG. 26 .

The SAM-expressing tau-CFP/tau-YFP biosensor cells without aggregates(Agg[−]) were transduced with a human genome-wide CRISPR hSAM sgRNAlibrary using a lentiviral delivery approach to transcriptionallyactivate each target gene. The sgRNAs in the library target sites within200 bp upstream of the transcription start site with an average coverageof ˜3 sgRNAs per gene. The sgRNAs were designed to avoid off-targeteffects by avoiding sgRNAs with two or fewer mismatches to off-targetgenomic sequences. The library covers 18,946 human genes. The librarywas transduced at a multiplicity of infection (MOI) <0.3 at a coverageof >300 cells per sgRNA. Tau biosensor cells were grown under zeocinselection to select cells with integration and expression of a uniquesgRNA per cell. Five independent screening replicates were used in theprimary screen.

At Day 10 (PM) post-transduction of the lentivirally-packaged CRISPRlibraries, the “maximal seeding” material was added to the cells, and atDay 13 (AM) FACS was performed to separate and collect FRET[−] andFRET[+] populations. See FIG. 26 . The screening consisted of fivereplicated experiments. DNA isolation and PCR amplification of theintegrated sgRNA constructs allowed a characterization by nextgeneration sequencing (NGS) of the sgRNA repertoire at each time point.For the “maximal seeding,” three amounts of lipofectamine were used permL of fresh medium (2.5 μL, 3.5 μL, and 4 μL), and different amounts ofcell lysate were tested per mL of fresh medium (3 μg, 4 μg, and 5 μg).

Day 13 FRET[−] and FRET[+] samples were analyzed for sgRNAs whoseenrichment or depletion may indicate an effect on the tau aggregationphenotype. sgRNAs that are enriched in Day 13 FRET[−] cells relative toDay 13 FRET[+] and Day 10, and/or are depleted in Day 13 FRET[+] cellsrelative to Day 13 FRET[−] and Day 10 indicate target genes that, whentranscriptionally activated, may reduce or protect against tauaggregation. These sgRNA hits are of special interest as potentialtargets for therapeutic intervention. sgRNAs that are enriched in Day 13FRET[+] cells relative to Day 13 FRET[−] and Day 10, and/or are depletedin Day 13 FRET[−] cells relative to Day 13 FRET[+] and Day 10, indicatetarget genes that, when transcriptionally activated, may promote orenhance tau aggregation. Data are analyzed and tested in secondaryscreens for validation as in Example 6. Statistical analysis mirrors theapproach used in Example 2. We compare (paired analysis) FRET(−) vsFRET(+) vs Day 10.

Example 8. Genome-Wide CRISPR/Cas9 Screening to Identify GeneticModifiers of Tau Disaggregation

In the above examples, we conducted two series of genome wide screensaiming at the identification of positive and negative regulators of tauaggregation. In our first screen, we have identified modifier geneswhich, when disrupted, promote the formation of tau aggregates whenstimulated with a weak tau seeding material. This “minimal-seeding”material consists of a conditioned medium collected from tau-YFPaggregate[+] cells, applied directly to 7B10C3 cells to trigger ˜0.1%FRET[+] cells after 3 Days In Vitro (DIV). In contrast, in the secondscreen we have treated our biosensor cells with a potent or maximal tauseeding material, that normally causes tau aggregation and FRETinduction in a majority of cells. This “maximal-seeding” materialconsists of sonicated whole cell lysate from tau-YFP aggregate[+] cells,applied with lipofectamine transfection reagent.

Here, we transduced aggregate[+] 7B10C3-B2 or DC11-B6 cells withlentivirally-packaged CRISPR libraries. 7B10C3-B2 and DC11-B6 are clonesthat contains stably propagating Tau aggregates. This screen was done inHEK293 tau biosensor cells expressing Cas9 (clone 7B10C3) or dCas9-SAM(clone DC11), in which tau aggregation produces a FRET signal that canbe detected and used as a means of cell sorting by FACS. The 7B10C3 andDC11 clones were further treated with tau fibrils in order to derivesub-clones that contains stably propagating Tau aggregates. Two of theseaggregate-positive aggregate[+] stable sub-clones, called 7B10C3-B2 andDC11-B6, were selected for expansion and used for screening.

After two weeks in culture, we isolated and sequenced by next generationsequencing (NGS) to reveal depleted/enriched single guide RNAs (sgRNAs)in FRET[−] cells on the hypothesis that sgRNAs that cause a loss of tauaggregation through disruption of their specific targets will beenriched in the FRET[−] population. The FRET[−] cell population waspredominantly composed by cells with no aggregates. However, we observedsome FRET[−] cells showing speckles, composed by tiny aggregates thatwere not sufficient to emit FRET and thus not recognized as FRET[+] byFACS.

In order to reduce false positives in the FRET[−] population and tominimize the number of speckles[+] cells observed predominately on G1phase after mitosis (see FIG. 28 ), we synchronized the cell cycleprogression by double thymidine block, a DNA synthesis inhibitor (seeFIG. 29 ). This novel application of this synchronization method allowedus to obtain a cell population predominantly enriched in S phase andthus to synchronize aggregate accumulation after mitosis.

Five replicate screens are performed for each library (two CRISPRnlibraries and one CRISPRa library). In each replicate, samples of7B10C3-B2 and DC11-B6 biosensor cells are collected at Day 7 and Day 10post-transduction of the lentivirally-packaged CRISPR libraries. At Day12, cells are grown in the presence thymidine for 21 hours. After thefirst block thymidine is removed (Day 13), cells are washed and grown infresh medium for 8 hours to release cells from the block and incubatedwith thymidine again for the second block. As a result of thissynchronization, cells progress synchronously through G2 and mitoticphase and are arrested at the beginning of S phase. At Day 14, cells arereleased by the second thymidine block and grown in fresh medium for 3hours to let them progress synchronously through G2 phase. FACS isperformed to separate and collect FRET[−] and FRET[+] populations. SeeFIG. 27 .

Genomic DNA is collected from each sample, and the sgRNA repertoireamplified from each by PCR. There are 60 samples in total: 5 replicatescreens*3 libraries*4 samples for each (Day 7, Day 10, Day 14 FRET[−],and Day 14 FRET[+].

Data analysis and statistical analysis mirror the approach used inExample 2, so the details from Example 2 are not repeated here. sgRNAsthat are enriched in Day 14 FRET[−] cells relative to Day 14 FRET[+],Day 7, and Day 10, and/or are depleted in Day 14 FRET[+] cells relativeto Day 14 FRET[−], Day 7, and Day 10, may indicate target genes that,when disrupted (or transcriptionally activated in the case of theCRISPRa screen), induce tau disaggregation. These sgRNA hits are ofspecial interest as potential targets for therapeutic intervention.sgRNAs that are enriched in Day 14 FRET[+] cells relative to Day 14FRET[−], Day 7, and Day 10, and/or are depleted in Day 14 FRET[−] cellsrelative to Day 14 FRET[+], Day 7, and Day 10, may indicate target genesthat, when disrupted (or transcriptionally activated in the case of theCRISPRa screen), promote or enhance tau aggregation.

We claim:
 1. A method of screening for genetic modifiers of tauaggregation, comprising: (a) providing a population of cells comprisinga Cas protein, a first tau repeat domain linked to a first reporter, anda second tau repeat domain linked to a second reporter, wherein thecells are mammalian cells, wherein the first reporter and the secondreporter are fluorescent proteins, and wherein the first reporter andthe second reporter are a fluorescence resonance energy transfer (FRET)pair; (b) introducing into the population of cells a library comprisinga plurality of unique guide RNAs that target a plurality of genes; (c)culturing the population of cells to allow genome editing and expansion,wherein the plurality of unique guide RNAs form complexes with the Casprotein, and the Cas protein cleaves the plurality of genes resulting inknockout of gene function to produce a genetically modified populationof cells; (d) contacting the genetically modified population of cellswith a tau seeding agent to produce a seeded population of cells,wherein step (d) comprises culturing the genetically modified populationof cells in the presence of conditioned medium harvested from culturedtau-aggregation-positive cells in which a tau repeat domain stablypresents in an aggregated state, wherein the conditioned medium washarvested after being on confluent tau-aggregation-positive cells for 1to 7 days; (e) culturing the seeded population of cells to allow tauaggregates to form, wherein aggregates of the first tau repeat domainand the second tau repeat domain form in a subset of the seededpopulation of cells to produce an aggregation-positive population ofcells; and determining abundance of each of the plurality of uniqueguide RNAs in the aggregation-positive population of cells identified instep (e) relative to the genetically modified population of cells instep (c), wherein enrichment of a guide RNA in the aggregation-positivepopulation of cells identified in step (e) relative to the culturedpopulation of cells in step (c) indicates that the gene targeted by theguide RNA is a genetic modifier of tau aggregation, wherein disruptionof the gene targeted by the guide RNA enhances tau aggregation.
 2. Themethod of claim 1, wherein the Cas protein is a Cas9 protein.
 3. Themethod of claim 2, wherein the Cas protein is Streptococcus pyogenesCas9, wherein the Cas protein comprises SEQ ID NO: 21 or wherein the Casprotein is encoded by a coding sequence comprising the sequence setforth in SEQ ID NO:
 22. 4. The method of claim 1, wherein the Casprotein, the first tau repeat domain linked to the first reporter, andthe second tau repeat domain linked to the second reporter are stablyexpressed in the population of cells and are genomically integrated inthe population of cells.
 5. The method of claim 1, wherein each guideRNA targets a 5′ constitutive exon or wherein each guide RNA targets afirst exon, a second exon, or a third exon.
 6. The method of claim 1,wherein step (c) is about 3 days to about 9 days.
 7. The method of claim6, wherein step (c) is about 6 days.
 8. The method of claim 1, whereinthe conditioned medium was harvested after being on confluenttau-aggregation-positive cells for about 4 days.
 9. The method of claim1, wherein step (d) comprises culturing the genetically modifiedpopulation of cells in about 75% conditioned medium and about 25% freshmedium.
 10. The method of claim 1, wherein the genetically modifiedpopulation of cells is not co-cultured with the tau-aggregation-positivecells in which a tau repeat domain stably presents in an aggregatedstate.
 11. The method of claim 1, wherein step (e) is about 2 days toabout 6 days.
 12. The method of claim 11, wherein step (e) is about 4days.
 13. The method of claim 1, wherein the aggregation-positivepopulation of cells in step (e) is identified by flow cytometry.
 14. Themethod of claim 1, wherein abundance is determined by next-generationsequencing.
 15. The method of claim 1, wherein a guide RNA is consideredenriched if the abundance of the guide RNA relative to the totalpopulation of the plurality of unique guide RNAs is at least 1.5-foldhigher in the aggregation-positive population of cells in step (e)relative to the cultured population of cells in step (c).
 16. The methodof claim 1, wherein step (f) comprises determining abundance of each ofthe plurality of unique guide RNAs in the aggregation-positivepopulation of cells in step (e) relative to the cultured population ofcells in step (c) at a first time point in step (c) and/or a second timepoint in step (c).
 17. The method of claim 16, wherein the first timepoint in step (c) is at a first passage of culturing the population ofcells, and the second time point is in the middle of culturing thepopulation of cells to allow genome editing and expansion.
 18. Themethod of claim 17, wherein the first time point in step (c) is afterabout three days of culturing, and the second time point in step (c) isafter about six days of culturing.
 19. The method of claim 16, wherein agene is considered a genetic modifier of tau aggregation, whereindisruption of the gene enhances tau aggregation, if: (1) the abundanceof a guide RNA targeting the gene relative to the total population ofthe plurality of unique guide RNAs is at least 1.5-fold higher in theaggregation-positive population of cells in step (e) relative to thecultured population of cells in step (c) at both the first time point instep (c) and the second time point in step (c); and/or (2) the abundanceof at least two unique guide RNAs targeting the gene relative to thetotal population of the plurality of unique guide RNAs is at least1.5-fold higher in the aggregation-positive population of cells in step(e) relative to the cultured population of cells in step (c) at eitherthe first time point in step (c) or the second time point in step (c).20. The method of claim 1, wherein the following steps are taken in step(f) to identify a gene as a genetic modifier of tau aggregation, whereindisruption of the gene enhances tau aggregation: (1) identifying whichof the plurality of unique guide RNAs are present in theaggregation-positive population of cells produced in step (e); (2)calculating the random chance of the guide RNAs identified in step(f)(1) being present using the formula nCn′*(x−n′)C(m−n)/xCm, wherein xis the variety of unique guide RNAs introduced into the population ofcells in step (b), wherein m is the variety of unique guide RNAsidentified in step (f)(1), wherein n is the variety of unique guide RNAsintroduced into the population of cells in step (b) that target thegene, and wherein n′ is the variety of unique guide RNAs identified instep (f)(1) that target the gene; (3) calculating average enrichmentscores for the guide RNAs identified in step (f)(1), wherein theenrichment score for a guide RNA is the relative abundance of the guideRNA in the aggregation-positive population of cells produced in step (e)divided by the relative abundance of the guide RNA in the culturedpopulation of cells in step (c), and wherein relative abundance is theread count of the guide RNA divided by the read count of the totalpopulation of the plurality of unique guide RNAs; and (4) selecting thegene if a guide RNA targeting the gene is significantly below the randomchance of being present and above a threshold enrichment score.
 21. Themethod of claim 1, wherein the first tau repeat domain and/or the secondtau repeat domain is a human tau repeat domain.
 22. The method of claim1, wherein the first tau repeat domain and/or the second tau repeatdomain comprises a pro-aggregation mutation, wherein the pro-aggregationmutation is a tau P301S mutation, which corresponds to position 59 ofSEQ ID NO:
 11. 23. The method of claim 1, wherein the first tau repeatdomain and/or the second tau repeat domain comprises a tau four-repeatdomain.
 24. The method of claim 1, wherein the first tau repeat domainand/or the second tau repeat domain comprises SEQ ID NO:
 11. 25. Themethod of claim 1, wherein the first tau repeat domain and the secondtau repeat domain are the same and each comprises a tau four-repeatdomain comprising a tau P301S mutation, which corresponds to position 59of SEQ ID NO:
 11. 26. The method of claim 1, wherein the first reporteris cyan fluorescent protein (CFP) and the second reporter is yellowfluorescent protein (YFP).
 27. The method of claim 1, wherein the cellsare human cells.
 28. The method of claim 27, wherein the cells areHEK293T cells.
 29. The method of claim 1, wherein the plurality ofunique guide RNAs are introduced at a concentration selected such that amajority of the cells receive only one of the unique guide RNAs.
 30. Themethod of claim 1, wherein the plurality of unique guide RNAs target 100or more genes, 1000 or more genes, or 10000 or more genes.
 31. Themethod of claim 1, wherein the library is a genome-wide library.
 32. Themethod of claim 1, wherein a plurality of target sequences are targetedon average in each of the targeted plurality of genes.
 33. The method ofclaim 32, wherein at least three target sequences are targeted onaverage in each of the targeted plurality of genes or wherein aboutthree to about six target sequences are targeted on average in each ofthe targeted plurality of genes.
 34. The method of claim 1, wherein theplurality of unique guide RNAs are introduced into the population ofcells by lentiviral transduction, wherein each of the plurality ofunique guide RNAs is in a separate viral vector.
 35. The method of claim34, wherein the population of cells is infected at a multiplicity ofinfection of less than 0.3.
 36. The method of claim 1, wherein theplurality of unique guide RNAs are introduced into the population ofcells together with a selection marker that imparts resistance to adrug, and step (b) further comprises selecting cells that comprise theselection marker.
 37. The method of claim 1, wherein the population ofcells into which the plurality of unique guide RNAs are introduced instep (b) comprises greater than 300 cells per unique guide RNA.